U.S. Department of the Interior

U.S. Geological Survey

Open File Report 2018–1096

Procedures for Using the Horiba Scientific Aqualog®

Fluorometer to Measure Absorbance and Fluorescence from 

Dissolved Organic Matter

AAXXXX_fig 01

A. Peat soil B. Rice

C. Cattail D. Algae

<0

<6

<12

<18

<24

<30

≥30

<0

<3

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≥15

<0

<3

<6

<9

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≥15

<0

<20

<40

<60

<80

<100

<120

≥120

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Emission, in nanometers

600

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Emission, in nanometers

600

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Emission, in nanometers

600

580

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Emission, in nanometers

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Cover. Four excitation-emission matrices showing fluorescence from different sources of dissolved organic matter common to

 the Sacramento–San Joaquin Delta (California, USA): A, Peat soil (euic, thermic Typic Medisaprists); B, Rice (oryza sativa);

C, Cattail (typha spp.); and D, Algae (thalassiosira weissflogii). Note, the presence or absence of fluorescence peaks,

 intensity of fluorescence response, changes in the ratios of excitation-emission pairs, and shifts in peak maxima have all been 

 shown to provide information about dissolved organic matter character and origin. 
Procedures for Using the Horiba Scientific 

Aqualog® Fluorometer to Measure 

Absorbance and Fluorescence from 

Dissolved Organic Matter

By Angela M. Hansen, Jacob A. Fleck, Tamara E.C. Kraus, Bryan D. Downing, 

Travis von Dessonneck, and Brian A. Bergamaschi

Open File Report 2018–1096

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

RYAN K. ZINKE, Secretary

U.S. Geological Survey

James F. Reilly II, Director

U.S. Geological Survey, Reston, Virginia: 2018

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Suggested citation:

Hansen, A.M., Fleck, J.A., Kraus, T.E.C., Downing, B.D., von Dessonneck, T., and Bergamaschi, B.A., 2018, Procedures 

for using the Horiba Scientific Aqualog® fluorometer to measure absorbance and fluorescence from dissolved organic 

matter: U.S. Geological Survey Open-File Report 2018–1096, 31 p., https://doi.org/10.3133/ofr20181096.

ISSN 2331-1258 (online)
iii

Contents

Abstract...........................................................................................................................................................1

Purpose and Scope .......................................................................................................................................1

Background.....................................................................................................................................................1

Sample Collection and Handling .................................................................................................................5

Analytical Method..........................................................................................................................................5

Aqualog® Instrument............................................................................................................................5

Equipment and Materials ....................................................................................................................6

Precautions and Interferences ..........................................................................................................6

Validation and Quality-Control Samples ...........................................................................................7

Monthly Quality Control ..............................................................................................................7

Aqualog® Validation Scans ...............................................................................................7

Potassium Dichromate.......................................................................................................8

Fluorescence Reference Set ............................................................................................8

Certified Reference Material ............................................................................................8

Daily Quality Control....................................................................................................................8

Standard Reference Material.........................................................................................11

Laboratory Blank Samples ..............................................................................................12

Laboratory Sample Replicates .......................................................................................12

Analysis Procedure ............................................................................................................................13

Data Reporting and Limits .................................................................................................................14

Acceptance of Data ...........................................................................................................................16

Data Processing and Corrections.............................................................................................................18

Data Storage.................................................................................................................................................18

Data Analysis................................................................................................................................................21

Summary........................................................................................................................................................25

Acknowledgments.......................................................................................................................................25

References Cited..........................................................................................................................................25

Appendix 1. Aqualog® Standard Operating Procedure Walkthrough ............................................31

Appendix 2. Processed Summary Report for Absorbance Data .....................................................31

Appendix 3. Processed Summary Report for Fluorescence Data...................................................31
iv

Figures

1. Schematics showing chromophoric dissolved organic matter components.....................2

2. Schematics showing some chromophoric dissolved organic matter components..........3

3. Graph showing the absorbance response for five concentrations of potassium 

dichromate solution averaged over an annual cycle.............................................................9

4. Graph showing absorbance response of five concentrations of potassium 

dichromate at three wavelengths measured over an annual cycle tests linearity 

through the ultraviolet range ......................................................................................................9

5. Graphs showing long-term average excitation-emission matrices for Starna 6BF 

PMMA (polymethylmethacrylate) blocks ...............................................................................10

6. Example showing excitation-emission matrix of the standard reference material 

SRMTea used in the Organic Matter Research Laboratory...................................................11

7. Graphs showing Aqualog® water Raman criteria.................................................................15

8. Graphs used for the visual inspection of the daily baseline excitation-emission 

matrix ............................................................................................................................................16

9. Graph showing absorbance spectra measured between 240 and 600 nanometers 

indicating the long-term method detection limit calculated using 1200 baselinecorrected blanks analyzed during January 2013–December 2014.....................................17

10. Graph showing fluorescence excitation-emission matrix indicating the long-term 

method detection limit calculated using 1200 baseline-corrected blanks analyzed 

January 2013–December 2014..................................................................................................17

11. Graphs showing example of model output from principal component analysis..............22

12. Graph showing an example of model output from discriminant analysis.........................23

13. Graphs showing an example of model output from parallel factor analysis....................24

Tables

1. Description of commonly used optical properties of absorbance and fluorescence 

to analyze the composition of dissolved organic matter .......................................................4

2. Laboratory equipment and materials used for the measurement of dissolved 

organic matter by absorbance and fluorescence in the U.S. Geological Survey 

Organic Matter Research Laboratory .......................................................................................6

3. Validation scans recommended for Aqualog® calibration ....................................................8

4. The National Institute of Standards and Technology recommended wavelengths 

and target results for five concentrations of potassium dichromate standard .................9

5. The scan order for a typical analysis run includes the water Raman, baseline, 

and analytical set........................................................................................................................12

6. Aqualog® experiments used in a typical analytical run, along with method, 

parameters, and reagents.........................................................................................................13

7. The Organic Matter Research Laboratory data-storage structure for example 

analysis date of May 18, 2016 ...................................................................................................19

8. National Water Information System codes for commonly published diagnostic 

parameters and indices.............................................................................................................20
v

Conversion Factors

International System of Units to U.S. customary units

By Multiply To obtain

Length

nanometer (nm) 25,400,000.0 inch (in.)

micrometer (μm) 25,400.0 inch (in.)

centimeter (cm) 2.54 inch (in.)

meter (m) 0.3048 foot (ft)

Volume

microliter (μL) 29,573.5 ounce, fluid (fl oz)

milliliter (mL) 29.5735 ounce, fluid (fl oz)

liter (L) 0.0295735 ounce, fluid (fl oz)

Flow rate

meter per second (m/s) 0.3048 foot per second (ft/s)

cubic meter per second (m3/s) 0.0283168 cubic foot per second (ft3/s)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F = (1.8 × °C) + 32.

Supplemental Information

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 

25 °C).

Concentrations of chemical constituents in water are given in either milligrams per liter (mg/L) 

or micrograms per liter (μg/L).
vi

Abbreviations

AU absorbance units

CAWSC California Water Science Center

CRM certified reference material

DA discriminant analysis

DBP disinfection byproduct

DDL daily detection limit

DOC dissolved organic carbon

DOM dissolved organic matter

EEM excitation-emission matrix

HNO3 nitric acid

IFE inner-filtering effects

IHSS International Humic Substances Society

LRW lab reagent water

LT-MDL long-term method detection limit

MDL method detection limit

NIST National Institute of Standards and Technology

NWIS U.S. Geological Survey National Water Information System database

ORML Organic Matter Research Laboratory

PARAFAC parallel factor analysis

PCA principal component analysis

PMMA polymethylmethacrylate

POM particulate organic matter

QA quality assurance

QC quality control

RPD relative percentage difference

RU Raman-normalized intensity units

SOP standard operating procedure

SRM standard reference material

USGS U.S. Geological Survey

UV ultraviolet
Procedures for Using the Horiba Scientific Aqualog®

Fluorometer to Measure Absorbance and Fluorescence 

from Dissolved Organic Matter

By Angela M. Hansen, Jacob A. Fleck, Tamara E.C. Kraus, Bryan D. Downing, Travis von Dessonneck, and 

Brian A. Bergamaschi

Abstract

Advances in spectroscopic techniques have led to an 

increase in the use of optical measurements (absorbance 

and fluorescence) to assess dissolved organic matter 

composition and infer sources and processing. Although 

optical measurements are easy to make, they can be affected 

by many variables rendering them less comparable, including 

by inconsistencies in sample collection (for example, filter 

pore size, preservation), the application of corrections for 

interferences (for example, inner-filtering corrections), 

differences in holding times, and instrument drift (for example, 

lamp intensity). A documented, standardized procedure to 

address these variables ensures that the optical (absorbance 

and fluorescence) measurements collected by U.S. Geological 

Survey researchers are useful and widely comparable.

Rigorous and quantifiable quality assurance and quality 

control are essential for making these data comparable, 

particularly because there is no published guideline for the 

measurement of dissolved organic matter absorbance and 

fluorescence, and especially because there is no National 

Institute of Standards and Technology standard for dissolved 

organic matter. Validation and quality-control samples are 

analyzed on a monthly basis to determine laboratory and 

instrument precision and daily (that is, each day samples are 

run) to ensure repeatability. Data are not considered acceptable 

unless they meet laboratory criteria: All standards should be 

within 10 percent of the target value, laboratory replicates 

should be within 5 percent relative percent difference, and 

laboratory blanks (that is, laboratory reagent-grade water) 

should be less than one-tenth of the long-term method 

detection limit.

Finally, for data to be useful, they must be accessible to 

users in a format that can be easily analyzed and interpreted. 

The Organic Matter Research Laboratory staff has developed 

a processing routine that extracts a subset of the data, which 

is made available to the public through the USGS National 

Water Quality Information System (http://nwis.waterdata.usgs.

gov/usa/nwis/qwdata), and organizes the full datasets (that 

is, complete absorbance spectra and fluorescence excitationemission matrices) in different forms that allow for these data 

to be analyzed using multi-parameter and multi-way statistical 

approaches.

Purpose and Scope

The purpose of this report is to document the procedures 

developed by the U.S. Geological Survey (USGS) California 

Water Science Center (CAWSC) Organic Matter Research 

Laboratory (OMRL) personnel for using the Aqualog®

fluorometer to measure the absorbance and fluorescence of 

dissolved organic matter (DOM). Topics include sample 

collection and handling, instrument set-up, data quality 

assurance and quality control, data processing, as well as a 

brief overview of optical properties and how these data are 

commonly used. The intended audience is primarily those 

who will use the Aqualog® fluorometer for research and could 

benefit from a guide to its specific operation following a set 

of standard procedures; as such, this report does not address 

the fundamental principles underlying the measurement 

technology.

Background

Optical spectroscopy has been used for decades to 

measure DOM amount and composition, and advances in 

instrumentation have improved data quality—and the ease of 

sample analysis—associated with this approach. The optical 

measurements discussed in this report can be broken up into 

two different phenomena: (1) absorbance, the measurement 

of the amount of light absorbed by a water sample over 

a known distance at a specific wavelength (fig. 1A), and 

(2) fluorescence, the measurement of the amount of light 

emitted by a water sample at a specific wavelength following 

absorbance of incident light over a known distance at a 

specific excitation wavelength (fig. 2A). Absorbance scans 
2 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

are depicted in a two-dimensional array, with wavelength, 

in nanometers (nm), on one axis and the amount of light 

absorbed on the second axis (fig. 1B). Fluorescence scans 

comprise sets of emission spectra collected across an array 

of excitation wavelengths and are typically depicted in 

an excitation-emission data matrix (EEM) consisting of 

thousands of excitation-emission pairs from a single water 

sample, where the excitation wavelength (nm) is on one axis, 

the emission wavelength (nm) is on the second axis, and the 

fluorescence intensity is on a third axis (fig. 2B).

Advances in commercially available optical spectroscopy 

instruments and analytical techniques have led to an increase 

in the use of optical property measurements of absorbance and 

fluorescence in many research applications across scientific 

disciplines. Optical spectroscopy increasingly not only is used 

to provide a proxy for DOM concentration and composition, 

but also to identify unique parameters that can be used to 

trace the DOM source, biogeochemical transformations, 

and roles in ecosystem processes (Jaffé and others, 2008; 

Hernes and others, 2009; Hansen and others, 2016). These 

improved techniques are rapid, inexpensive, and allow for 

tracking DOM both in surface and groundwater. Optical 

measurements have been used in a range of aquatic systems 

(reservoirs, streams, estuaries, oceans) as well as to identify 

DOM associated with plants, soil, phytoplankton, wastewater, 

oil, and other materials. Optical data can be used in a wide 

range of applications for natural waters, including organic 

matter cycling (Coble, 2007; Tranvik and others, 2009), algal 

production of DOM (Lapierre and Frenette, 2009), and DOM 

source attribution and fingerprinting (Baker and Spencer, 

2004; Carstea and others, 2009; Goldman and others, 2012; 

Carpenter and others, 2013). 

B

A254

Spectral slope

Intensity, in absorbance units 

1.0

0.8

0.6

0.4

0.2

0

200 250 300 350 400 450

Wavelength, in nanometers

500 550 600 650 700

A

EXPLANATION

Sample 1

Sample 2

Sample 3

Figure 1. Chromophoric dissolved organic matter (DOM) components: A, absorb light thereby decreasing the amount of energy 

exiting the sample and B, the absorbance response at a single wavelength is related to DOM concentration (that is, absorbance 

at 254 nanometers [A254]), whereas the slope between two wavelengths or the ratios between them provides information about the 

composition of the DOM.
Background 3

Measuring DOM absorbance and fluorescence 

in a filtered-water sample provides information about 

the concentration of the bulk DOM pool as well as the 

composition of different types of compounds present and their 

likely origin. Common parameters and indices derived from 

optical data include the absorbance at specific wavelengths 

(for example, absorbance at 254, 280, 370, 412, and 440 nm) 

and fluorescence at specific excitation-emission pairs (for 

example, ex260/em450, peak A; ex275/em340, peak T; and 

ex340/em440, peak C). The response at a specific wavelength 

or wavelength pair is related to the DOM concentration—

the response increases as the amount of the optically active 

DOM pool in the sample increases. Information about the 

composition of DOM can be obtained by normalizing the 

absorbance or fluorescence response to another parameter; 

normalizing to dissolved organic carbon (DOC) concentration 

is most commonly reported (Beggs and Summers, 2011; 

Hansen and others, 2016). In table 1, various indicators of 

DOM composition are listed, including the examination of 

ratios of different wavelengths (for example, fluorescence 

index and humification index) and spectral slopes (S275–295, 

S290–350, S350–400) across specific regions of the optical spectrum, 

which can be related to the molecular weight, source, and 

processing (for example, biodegradation and photolytic 

exposure) of DOM.

sac17-0642_fig02

AEmission, in nanometers

Emission, in nanometers

Excitation, in nanometers

600 0.22

0.02

0.18

0.16

0.14

0.12

0.01

0.08

0.06

0.04

0.02

0.01

0.08

0.06

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0.02

600

550

500

450

350

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450

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400

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Intensity, in Raman units

Intensity, in Raman units

0.01

0.08

0.06

0.04

0.02

0

240 260 280 300 320 340 400 420 440

Excitation, in nanometers

360 380

B

C

Intensity, in Raman units

A

Figure 2. Some chromophoric dissolved organic matter (DOM) 

components: A, absorb light then re-emit it at a longer wavelength 

and B, a two-dimensional representation of fluorescence is 

referred to as an excitation-emission matrix (EEM) plot. As with 

absorbance, the response at any given wavelength can be related 

to concentration; C, an EEM is actually a three-dimensional 

surface with the z-axis indicating intensity.
4 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

Table 1. Description of commonly used optical properties of absorbance and fluorescence to analyze the composition of dissolved 

organic matter.

[Adapted from Hansen and others, 2016. Abbreviations: cm, centimeter; DOC, dissolved organic carbon; DOM, dissolved organic matter; ex-em, excitationemission; FDOM, fluorescent dissolved organic matter; L, liter; mg, milligram; nm, nanometer; RU, Raman units; S, slope; SUVA, specific ultraviolet 

absorbance; UV, ultraviolet; VIS, visibile; >, greater than; —, not applicable]

Measurements Description 

Calculation Purpose Reference

Absorbance measurements

SUVA at 254 nm, in liters per 

milligram of carbon per meter

Absorption coefficient at 254 nm 

divided by DOC concentration.

Absorbance per unit carbon. 

Typically a greater number is 

associated with greater aromatic 

content.

Weishaar and others 

(2003); Spencer 

and others (2012); 

Chowdhury (2013) 

SUVA (280 nm, 350 nm, 370 nm)

in liters per milligram of 

carbon per meter

Absorption coefficient at a given 

wavelength in the ultraviolet region 

divided by DOC concentration.

Absorbance per unit carbon. 

Typically a greater number is 

associated with greater aromatic 

content.

Chin and others (1994); 

Hansen and others 

(2018)

Specific visible absorbance 

[SVA (412 nm, 440 nm, 

480 nm, 510 nm, 532 nm, 

555 nm)] in liters per 

milligram of carbon per meter

Absorption coefficient at a given 

wavelength in the visible region 

divided by DOC concentration.

Absorbance per unit carbon. 

Typically a greater number is 

associated with greater aromatic 

content.

Hansen and others 

(2016)

Spectral slopes 

(S275–295, S290–350, S350–400)] (nm–1)

Nonlinear fit of an exponential 

function to the absorption spectrum 

over the wavelength range.

Typically higher S values indicate 

low molecular weight material and 

(or) decreasing aromaticity.

Blough and Del Vecchio 

(2002); Helms and 

others (2008)

Spectral slope ratio (SR) 

S275–295 (nm–1):S350–400 (nm–1)

Spectral slope S275–295 divided by 

spectral slope S350–400.

Shown to be negatively correlated 

to DOM molecular weight 

and to generally increase upon 

irradiation.

Helms and others 

(2008)

Fluorescence measurements

Specific fluorescence at various 

peaks 

[spA, spB, spC, spD, spM, 

spN, spT, spZ](RU L mg-C–1)

Fluorescence at a given ex-em pair 

divided by DOC concentration.

Fluorescence per unit carbon. Hansen and others 

(2016, 2018)

Peak ratio (A:T) The ratio of Peak A (ex260/em450) to 

Peak T (ex275/em340) intensity.

An indication of the amount of 

humic-like (recalcitrant) versus 

fresh-like (labile) fluorescence in 

a sample.

Hansen and others 

(2016, 2018)

Peak ratio (C:A) The ratio of Peak C (ex340/em440) to 

Peak A (ex260/em450) intensity.

An indication of the relative amount 

of photosensitive humic-like DOM 

fluorescence in a sample.

Moran and others 

(2000); Hansen and 

others (2016)

Peak ratio (C:M) The ratio of Peak C (ex340/em440) to 

Peak M (ex300/em390) intensity.

An indication of the amount of 

diagenetically altered (blueshifted) fluorescence in a sample.

Coble (1996); Burdige 

and others (2004); 

Para and others 

(2010); Helms and 

others (2013)

Peak ratio (C:T) The ratio of Peak C (ex340/em440) to 

Peak T (ex275/em340) intensity.

An indication of the amount of 

humic-like (recalcitrant) versus 

fresh-like (labile) fluorescence in 

a sample.

Baker and others (2008)

Fluorescence index (FI) The ratio of em wavelengths at 

470 nm and 520 nm, obtained at 

ex 370.

Shown to identify the relative 

contribution of terrestrial and 

microbial sources to the DOM 

pool.

McKnight and others 

(2001); Cory and 

others (2010)
Analytical Method 5

Sample Collection and Handling

Samples processed in the OMRL are collected according 

to the USGS protocols (U.S. Geological Survey, 2012). 

It should be noted that the use of methanol to rinse field 

equipment and tubing results in contamination, so its use 

should be avoided when collecting samples for organic carbon 

analysis. After collection, samples are filtered immediately 

in the field or placed on ice and filtered as soon as possible 

(preferably within 24 hours) in the laboratory through precombusted 0.3-micrometer (μm) nominal pore-size glass-fiber 

filters (Advantec MFS model GF7547mm; Advantec MFS, 

Dublin, California, USA; also available through the USGS 

One-Stop Shop, item Q355FLD) or 0.45-μm nominal poresize syringe, capsule, or disk filters. Following filtration, 

samples in amber glass should be stored in the dark on ice or 

refrigerated at less than 4 degrees Celsius (°C) and analyzed 

within 2 days, preferably. Acid preservation of samples is 

not recommended. If samples are known to include a large 

fraction of labile DOM, it is best to filter samples immediately 

in the field and analyze them within 24 hours to avoid loss 

of that pool of DOM prior to analysis. Samples are visually 

inspected just prior to analysis to ensure no colloids or 

precipitates have formed during storage. In cases where 

samples become visually cloudy, refiltration may be required. 

Procedures for handling samples that are known to contain a 

large amount of volatile organic compounds (for example, oil 

and gas compounds) are being developed.

Analytical Method

In this section, everything needed to measure absorbance 

and fluorescence in a water sample is described, from the 

instrument and software to assessment of quality control and 

reporting of the data.

Aqualog® Instrument

The method used in the CAWSC OMRL is appropriate 

for using the Aqualog® instrument to analyze filtered-water 

samples containing DOM. Procedures for operating the 

Aqualog® are adapted from the manufacturer’s user manual 

(Horiba Scientific, 2011). This method simultaneously 

measures the absorbance and fluorescence of a filtered-water 

sample and produces an absorbance scan of 121 (361 after 

interpolation) wavelengths and a fluorescence matrix of 

11,029 discrete excitation and emission pairs.

Measurements Description 

Calculation Purpose Reference

Fluorescence measurements—Continued

Humification index (HIX) The area under the em spectra 

435–480 nm divided by the peak 

area 300–345 nm + 435–480 nm, 

at ex 254 nm.

An indicator of humic substance 

content or extent of humification. 

Higher values indicate an 

increasing degree of humification.

Ohno (2002)

Freshness index (β:α) The ratio of emission intensity at 

380 nm divided by the maximum 

emission intensity between 420 

and 435 nm at excitation 310 nm.

An indicator of recently produced 

DOM, with greater values 

representing a greater proportion 

of fresh DOM.

Parlanti and others 

(2000); Wilson and 

Xenopoulos (2009)

Relative fluorescence efficiency 

(RFE) (RU cm)

Ratio of fluorescence at ex370/em460 

(FDOM) to absorbance at 370 nm.

RFE is an indicator of the relative 

amount of algal and non-algal 

DOM.

Downing and others 

(2009)

Biological index (BIX) The ratio of emission intensity at 

380 nm divided by 430 nm at 

excitation 310 nm.

An indicator of autotrophic 

productivity. Greater values (>1) 

correspond to recently produced 

DOM of autochthonous origin.

Huguet and others 

(2009)

Table 1. Description of commonly used optical properties of absorbance and fluorescence to analyze the composition of dissolved 

organic matter.—Continued

[Adapted from Hansen and others, 2016. Abbreviations: cm, centimeter; DOC, dissolved organic carbon; DOM, dissolved organic matter; ex-em, excitationemission; FDOM, fluorescent dissolved organic matter; L, liter; mg, milligram; nm, nanometer; RU, Raman units; S, slope; SUVA, specific ultraviolet 

absorbance; UV, ultraviolet; VIS, visibile; >, greater than; —, not applicable]
6 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

Equipment and Materials

The equipment and materials listed in table 2 are for the 

measurement of absorbance and fluorescence of dissolved 

organic matter in waters using the Aqualog® instrument. The 

analyses are done in a lab free of organic solvents to prevent 

vapor-phase contamination of the samples. Type I (18.2 megaohm resistance, organic-free) laboratory reagent-grade 

water (LRW) is produced onsite with a recirculating water 

system (Labconco WaterPro Polishing System, Kansas City, 

Missouri). Routine replacement of water-polishing system 

cartridges and system sanitization is performed annually. All 

glassware is acid-washed in 10-percent weight-to-volume 

(wt/vol) nitric acid (HNO3)(aq) and water to minimize organic 

contamination.

The Aqualog® light source is a 150 watt ozone-free xenon 

arc lamp. Excitation and absorbance spectra are scanned with 

a double-grating monochrometer from 230 to 600 nm, with 

a 5-nm bandpass, in 3-nm increments, and emission spectra 

are collected with a charge-coupled device (CCD) from 250 

to 600 nm, with a 5-nm bandpass, in 1.6–3.3-nm (4-pixel) 

increments. Aqualog® instruments are equipped with different 

specifications for light sources and CCDs, both of which can 

affect the matrix size and resolution. Consideration should 

be given to the specific specifications of the Aqualog® when 

establishing laboratory-specific methods and tools. 

Precautions and Interferences

Ambient light interferes with measurements. As a 

result, the instrument is operated with the cover closed to 

prevent light from entering the chamber during instrument 

measurement.

All glassware should be cleaned meticulously. The 

OMRL procedure requires all glassware is washed with 

detergent and hot tap water. Glassware is then acid-washed 

by soaking in a 10 percent HNO3(aq) bath for at least 2 hours. 

Following the acid wash, the glassware is triple-rinsed 

inside and out with Type I LRW and dried on a laminar-flow 

table. Clean, dry glassware is then sealed by wrapping with 

aluminum foil and stored in a dust-free cabinet.

Laboratory water systems have been known to 

contaminate samples as a result of bacterial breakthrough 

from resin beds, activated carbon, and filters. Laboratory 

water systems should be maintained and monitored frequently 

for background carbon and bacterial growth. The OMRL 

procedures for maintaining clean LRW were described 

previously in the “Equipment and Materials” section of 

this report, and monitoring of this water as part of the daily 

quality management of an analytical run is explained later in 

this report.

Table 2. Laboratory equipment and materials used for the measurement of dissolved organic matter by absorbance and fluorescence 

in the U.S. Geological Survey (USGS) Organic Matter Research Laboratory.

[Equivalent products can be found elsewhere. Abbreviations: cm, centimeter; LRW, lab reagent water; Mohm, mega-ohm; mL, milliliter; n/a, not applicable; 

ppb, parts per billion; W, watt; w/w, weight per weight; U.S.A., United States of America; μL, microliter; μm, micrometer; %, percent; ±, plus or minus; 

<, less than]

Equipment and materials Manufacturer Part number

Acid, nitric (HNO3, 68–70% w/w) Fisher Scientific A200C-212

Aqualog® lamp (xenon, 150 W ozone free XBO) Horiba Scientific, New Jersey, U.S.A. 1905-OFR

Aqualog® instrument Horiba Scientific, New Jersey, U.S.A. n/a

Balance, certified accuracy of 0.050 gram±0.0001 n/a n/a

Bottles, amber glass, baked (40 mL, 125 mL, 250 mL) USGS One-Stop Shop N1560, Q28FLD, Q435FLD

Cuvettes (10-cm pathlength) Starna Cells, Inc. 3-Q-10

Filter (precombusted 47-mm diameter, 0.3‐μm nominal pore-size 

glass-fiber filters or 0.45-μm syringe filter)

USGS One-Stop Shop Q355FLD

Lens paper Fisher Scientific 11-997

Pipette (100–1,000 μL) Eppendorf 13-690-032

Pipette (1–10 mL) Eppendorf 13-690-034

Standard reference material (SRM) See “Validation and Quality-Control 

Samples” section in report text.

n/a

Type 1 LRW (18.2 Mohm resistance, <10 ppb total organic carbon) Labconco WaterPro PS 9000500

Workstation installed with Aqualog® software (version 3.6) n/a n/a
Analytical Method 7

Blemishes on the cuvette are a regular source of 

contamination and interference. These include manufacturing 

defects, scratches, fingerprints, oils, and stains that can 

cause buildup of optically active material and lead to the 

misrepresentation of detection limits and measurements. 

Always rinse the cuvette between each sample with plenty of 

Type I LRW at the source, always wear gloves, and replace 

gloves whenever in doubt of cleanliness. Confirm the cuvette 

is free from blemishes first by visual inspection and then by 

evaluation of the laboratory blanks (see later) in the analytical 

run. In the OMRL, lens paper is used to buff and prepare the 

cuvette for analysis.

Optical measurements are extremely sensitive to light 

scattering of fine particulates and colloids. Dissolved organic 

matter is operationally defined as the organic matter fraction 

that passes through a filter (typically 0.3–0.7 μm), and that 

which is collected on the filter is defined as particulate organic 

matter (POM). Although filter pore size typically does not 

affect DOM concentration, under some conditions, sorption 

and the formation of colloids and even precipitation can 

transfer DOM to the POM pool. Baker and others (2007) 

suggest filter pore size unevenly affects optical measurements 

and emphasize the need to standardize filter size in individual 

studies. Consideration of the effects of pore size should 

be determined prior to interpretation or comparison with 

published results. Larger or smaller pore sizes may be used 

in cases where optical measurements are compared to other 

analyses on the same water sample (for example, biological 

oxygen demand, chlorophyll-a concentration, disinfection 

byproduct formation); however, the user should be aware of 

potential interferences from colloids or particles. The filter 

pore size should be reported for all samples analyzed.

Several studies have looked at different approaches 

for storing samples including, for example, freezing or 

acidifying. In the OMRL, samples are filtered and analyzed 

as soon as possible, preferably within 2 days of sample 

collection. Freezing has been used to store organic-rich 

samples with varying degrees of success that depend on 

the initial DOC concentrations (Spencer and others, 2007; 

Fellman and others, 2008; Hudson and others, 2009), with 

DOC concentrations greater than 5 milligrams per liter (mg/L) 

and specific ultraviolet absorbance at 254 nm (SUVA254) 

values greater than 3.5 liters per milligram carbon per meter 

(L mg C–1 m–1) exhibiting greater loss of concentration as a 

result of precipitation and changes in chemical composition 

than samples with lower concentrations. In certain cases 

where DOC concentrations are low, freezing can be a 

practical storage method (Fellman and others, 2008); 

however, the effects of freezing and thawing or acidifying 

samples for preservation should be evaluated on a site- and 

study-specific basis.

Validation and Quality-Control Samples

Validation and quality-control samples are run on 

a monthly basis to determine laboratory and instrument 

precision and on a daily basis (that is, each day samples 

are run) to ensure repeatability. Ideally an official National 

Institute of Standards and Technology (NIST) certified 

reference material (CRM) would be used to determine 

the accuracy and precision of optical data across the full 

absorbance and fluorescence spectra, but such a standard for 

the complex composition of natural DOM does not exist. 

The available CRMs are limited to a specific fluorescence 

excitation-emission pair or absorbance region (for example, 

Starna quinine sulfate reference set, RM-4QS00, and Starna 

6BF fluorescence reference cells) and are therefore not 

informative enough to use on a daily basis. Furthermore, 

although the Starna quinine sulfate reference set (RM-4QS00) 

is available at a range of concentrations (0.25–1.0 mg/L), even 

the lowest concentration does not provide an environmentally 

relevant optical response. To address analytical quality 

assurance (QA), the OMRL staff measures commonly used 

optically active compounds monthly in coordination with 

daily standard reference materials to examine individual 

wavelengths and wavelength pairs, which we describe in detail 

in following sections.

Monthly Quality Control

Validation and quality-control samples are analyzed 

on a monthly basis to determine laboratory and instrument 

precision.

Aqualog® Validation Scans

Aqualog® validation scans are performed in the OMRL 

on a monthly basis to validate instrument performance. 

We perform four validation scans recommended by the 

manufacturer: (1) excitation validation, (2) water Raman SNR 

(signal-to-noise ratio) and emission calibration, (3) absorbance 

photometric accuracy, and (4) quinine sulfate unit. The 

purpose of the validation scans, the required materials, 

and criteria for a satisfactory response are summarized in 

table 3 and detailed further in the user’s manual (Horiba 

Scientific, 2011).
8 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

Potassium Dichromate

The use of potassium dichromate (K2Cr2O7) dissolved in 

dilute perchloric acid is a common method for validating the 

accuracy of the absorbance response (fig. 3) and the linearity 

of the concentration response (fig. 4) of a spectrophotometer 

in the ultraviolet (UV) region. In the OMRL, absorbance 

of potassium dichromate is measured at three wavelengths 

(that is, 257, 313, and 350 nm) specified by NIST. Figures 3

and 4 show data collected over an annual cycle that, for ease 

of comparison, are presented in a similar format to those in 

the Starna Cells (2015) guide for the NIST traceable UV/

Vis/NIR reference sets. These data are generated using the 

NIST traceable set (Starna Cells, Inc., Atascadero, Calif., part 

no. RM-0204060810) that contains five sealed cuvettes of 

potassium dichromate in a range of concentrations (table 4) 

and a cuvette containing perchloric acid (0.001 molar) 

to be used as a blank. Although NIST recommends also 

measuring absorbance at 235 nm, the Aqualog® method for the 

simultaneous collection of fluorescence and absorbance data 

begins scanning at wavelength 240 nm; therefore, we report 

results at 257, 313, and 350 nm.

Fluorescence Reference Set

Starna 6BF fluorescence reference cells are a set of 

seven fluorescing materials (anthracene and naphthalene, 

ovalene, p-terphenyl, tetraphenylbutadiene, compound 610, 

and rhodamine) set in six polymethylmethacrylate (PMMA) 

blocks in the dimensions of a standard cuvette. Each block has 

a unique excitation and emission curve, which allows the user 

to check the accuracy of the instrument’s response across a 

specified spectrum (fig. 5). Although this set is beneficial for 

targeting specific peaks (excitation and emission, or ex-em, 

pairs), it is limited by its inability to simultaneously provide 

a response both in the regions indicating more humic-like, 

recalcitrant material (peaks A, C) and the regions indicating 

the fresher, more labile material (peaks B, T) in a single scan.

Certified Reference Material

Certified reference materials (CRMs) are used to track 

accuracy and precision among analytical runs; however, there 

are few certified fluorescence standards available to properly 

validate instrument performance. The International Humic 

Substances Society (IHSS) standards are an option; however, 

preparing these to an exact concentration poses challenges 

because organic matter is not readily or consistently brought 

back into solution once fully dried (Mobed and others, 1996). 

Along with the fact IHSS standards are in limited supply, 

and no guidance documents exist for their preparation as 

a fluorescence standard material, they are also costly and, 

therefore, are not practical for frequent use.

Daily Quality Control

Validation and quality-control samples are analyzed 

on a daily basis (that is, each day samples are run) to ensure 

repeatability.

Table 3. Validation scans recommended for Aqualog® calibration.

[cps, counts per second; CCD, charge-coupled device; EEM, excitation-emission matrix; LRW, lab reagent water; mg/L, milligram per liter; nm, nanometer; 

QS, quinine sulfate; SNR, signal-to-noise ratio; SRM, standard reference material; >, greater than; ±, plus or minus]

Validation scan Purpose Required reagent/cell Satisfactory response

Excitation validation The purpose of this scan is to 

verify lamp performance and 

peak position.

This is a lamp scan. No cell or 

reagent required. Leave cell 

chamber empty.

Peak position 467 (±1 nm).

Water Raman SNR and 

emission calibration

This validation check examines 

the wavelength calibration 

of the CCD detector. It is 

an emission scan of the 

Raman-scatter band of water 

performed in right-angle 

mode. 

Standard cuvette with Type 1 

LRW.

Raman peak position 397 nm (±1 nm). 

The results column should read 

PASS. Select the tab “Raman SNR 

Calculation.” In the B(Y) column 

below the comments section, there 

should be a number. This is the lamp 

intensity, and the value should be 

>650,000 cps.

Absorbance photometric 

accuracy

This validation check examines 

the accuracy of the absorption 

function of the Aqualog®.

Standard SRM 935a (potassium 

dichromate blank, potassium 

dichromate 60 mg/L).

Select the tab “Test Results,” the “Pass/

Fail” column should display “P” for all 

four wavelengths measured.

Quinine sulfate unit 

(QSU)

This function provides a 

standardized intensity for 

fluorescence measurements 

and EEMs.

Quinine sulfate standard kit 

(RM-QS00) containing blank 

and standard (1 mg QS/L).

Select the tab “QSU Calculation.” The 

absorbance value at 347.5 nm should 

be 0.01384. Select the tab “Emission 

Spectrum Graph.” Using the crosshairs 

icon locate the position of the peak. 

This value should be 450 nm (±1 nm).
Analytical Method 9

sac17-0642_fig03

Concentrations in milligrams per liter

100

80

60

40

20

EXPLANATION

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Intensity, in absorbance units 

240 260 280 300 320 340 360 380 400 420 440

Wavelength, in nanometers

Figure 3. The absorbance response for five concentrations of potassium dichromate solution averaged over an annual cycle (sample 

count is 11). Peaks and trough are tracked at 257, 313, and 350 nanometer (nm) wavelengths. Error bars depicting standard deviation 

were less than 0.002 at each point and thus are not visible.

Wavelength in nanometers

257 313 350

EXPLANATION

Intensity, in absorbance units 

0 20 40 60 80 100

Potassium dichromate concentration, in milligrams per liter

R2=0.99

R2=0.99

R2=0.99

0

0.2

0.4

0.6

0.8

1.0

1.6

1.2

1.4

R-squared = coefficient of determination

Figure 4. Absorbance response of five concentrations of potassium dichromate at three wavelengths measured over an annual cycle 

(sample count is 11) tests linearity through the ultraviolet (UV) range. The error bars depicting standard deviation were less than 0.002 

absorbance units at each point and thus are not visible.

Table 4. The National Institute of Standards and Technology 

(NIST) recommended wavelengths and target results for five 

concentrations of potassium dichromate standard.

[mg/L, milligram per liter; nm, nanometer]

Concentration 257 nm 313 nm 350 nm

20 mg/L 0.281 0.095 0.209

40 mg/L 0.572 0.192 0.426

60 mg/L 0.862 0.289 0.634

80 mg/L 1.159 0.385 0.853

100 mg/L 1.448 0.480 1.069
10 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

sac17-0642_fig05

FDOM

HIX

FI

C

A

B

T

M

D

N

Z

β:α

O

FDOM

HIX

FI

C

A

B

T

M

D

N

Z

β:α

O

FDOM

HIX

FI

C

A

B

T

M

D

N

Z

β:α

O

FDOM

HIX

FI

C

A

B

T

M

D

N

Z

β:α

O

FDOM

HIX

FI

C

A

B

T

M

D

N

Z

β:α

O

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

A

C

E

FDOM

HIX

FI

C

A

B

T

M

D

N

Z

β:α

O

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

Excitation, in nanometers

EXPLANATION

Intensity, in Raman units

0

10

20

30

40

50

50

<

<

<

<

<

<

≥

EXPLANATION

Intensity, in Raman units

0

100

200

300

400

400

<

<

<

<

<

≥

EXPLANATION

Intensity, in Raman units

0

10

20

30

40

50

50

<

<

<

<

<

<

≥

EXPLANATION

Intensity, in Raman units

0

10

20

30

40

50

50

<

<

<

<

<

<

≥

EXPLANATION

Intensity, in Raman units

0

10

20

30

40

50

50

<

<

<

<

<

<

≥

EXPLANATION

Intensity, in Raman units

0

10

20

30

40

50

50

<

<

<

<

<

<

≥

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

B

D

F

Emission, in nanometers

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

Emission, in nanometers

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

Emission, in nanometers

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

Emission, in nanometers

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

Emission, in nanometers

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

Emission, in nanometers

Figure 5. Long-term average excitation-emission matrices (EEMs) for Starna 6BF PMMA (polymethylmethacrylate) blocks: 

A, anthracene and napthalene; B, ovalene; C, p-terphenyl; D, tetraphenyl butadeine; E, compound 610; and F, rhodamine. Each plot 

shows the average of 18 scans over a 22-month period. (Labeled regions of the EEM correspond to named peaks or table 1 optical 

properties; FDOM, fluorescent dissolved organic matter peak.)
Analytical Method 11

Standard Reference Material

The standard reference material (SRM) used in the 

OMRL for tracking accuracy and precision among analytical 

runs is a readily available, consumer-grade bottled tea (Pure 

Leaf®, unsweetened black tea, Purchase, New York), referred 

to as SRMTea in this report. Prior to use, the SRMTea is diluted 

to 1 percent concentration (1 milliliter of tea to 99 milliliters 

of Type I LRW); at this concentration, the known standard 

meets the requirement of inner-filtering corrections. This 

product has been proven to be stable and internally traceable 

over a long time (4 years) for absorbance and fluorescence 

spectra over the full range of measurement (fig. 6). Although 

minor variability exists among production lots (batches of tea), 

the different lots have been verified to meet the laboratory 

quality-assurance and quality-control (QA/QC) criteria across 

the relevant absorbance spectra and fluorescence EEMs to 

verify proper lamp function and processing consistency as 

an internal lab SRM. Users should take into account this is a 

consumer-grade food product, and although relatively stable, 

variability in the absorbance spectra and fluorescence EEMs 

relating to seasonal harvesting and source locations of teas has 

been observed during the 4-year tracking period.

Emission, in nanometers

Excitation, in nanometers

240 260 280 300 320 340 360 380 400 420 440

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

FDOM

HIX

FI

C

A

B

T

M

D

N

Z

β:ɑ

EXPLANATION

0

20

40

60

80

100

100

<

<

<

<

<

<

≥

Intensity, in Raman units

Figure 6. Excitation-emission matrix (EEM) of the standard reference material (Pure Leaf®, unsweetened black tea, Purchase, 

New York) SRMTea used in the Organic Matter Research Laboratory. The EEM shows the long-term average for SRMTea, which includes 

189 scans from 22 lot numbers over a 12-month period. (Labeled regions of the EEM correspond to named peaks or table 1 optical 

properties; FDOM, fluorescent dissolved organic matter peak.)
12 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

Although only a small amount of an 8 ounce (oz) bottle 

of SRMTea is needed to prepare a standard, each bottle is used 

only for a maximum of 1 month after opening, with at least 

1 week of overlap between old and new bottles. The OMRL 

lab staff analyzes two different bottles in each analytical run 

to verify internal consistency before using them as an SRM. 

Each bottle receives a unique laboratory identifier, and the lot 

number is recorded for long-term tracking purposes. Although 

full EEMs are collected for evaluation of performance, 

internal lab evaluation is tracked for 10 absorbance 

wavelengths and 8 widely published diagnostic fluorescence 

ex-em pairs. Acceptance is then based on recovery percentage 

(plus or minus 20 percent) of the long-term average of the 

SRM record.

Laboratory Blank Samples

Laboratory blank samples, or blanks, can reveal 

background levels and possible contamination of the 

equipment used during the analytical procedure (for example, 

cuvette blemishes, improper cuvette rinsing, LRW system 

quality control). A minimum of 5 laboratory blanks are 

measured in a typical analytical run; 3 blanks are run prior 

to running samples, and 2 blanks are run following each set 

of approximately 10 samples (table 5). Analyzing multiple 

baseline-corrected laboratory blanks daily allows the analyst 

to track blank-water quality and instrument drift during the 

course of the day. Individual blank results are acceptable 

when absorbance and fluorescence are less than one-tenth of 

the long-term method detection limit (LT-MDL). Calculating 

the standard deviation of all blanks in a single analytical 

run confirms the daily detection limit (DDL) is below the 

LT-MDL. Although the DDL is not a true detection limit, it 

serves as a diagnostic tool for daily contamination bias.

Laboratory Sample Replicates

Analysis of laboratory sample replicates (aliquots of the 

same sample) are a demonstration of precision as measured 

by the relative percent difference (RPD) between the 

measurements of the original sample and the replicate sample, 

calculated as 100 times the absolute difference between the 

two measurements divided by their average. Laboratory 

replicates not only check instrument precision, but also 

reflect the homogeneity of the sample and the precision of the 

analyst. A minimum of 1 laboratory replicate is measured in 

a typical analytical run (table 5), where 1 laboratory replicate 

is measured approximately every 10 samples. Laboratory 

replicates are considered acceptable when the RPD is less than 

5 percent.

Table 5. The scan order for a typical analysis run includes the 

water Raman, baseline, and analytical set.

[ba, baseline; bl, blank; MMDDYY, month day year; OMRL, Organic Matter 

Research Laboratory; SRM, standard reference material; —, not applicable]

Scan 

order

Sample 

type

File-naming 

convention Comments

1 Water Raman wrMMDDYY —

2 Baseline baMMDDYY —

3 Lab blank blMMDDYYa —

4 Lab blank blMMDDYYb —

5 Lab blank blMMDDYYc —

6 Sample OMRL 1 Sample names should be 

unique to the analyzing 

lab.

7 Sample OMRL 2 —

8 Sample OMRL 3 —

9 Sample OMRL 4 —

10 Sample OMRL 5 —

11 Sample OMRL 6 —

12 Sample OMRL 7 —

13 Sample OMRL 8 —

14 Sample OMRL 9 —

15 Sample OMRL 10 —

16 SRMTea SRMs01 Tea at 1-percent 

concentration. Analyze 

two SRMs once per 

day.

17 SRMTea SRMs01 Tea at 1-percent 

concentration. Analyze 

two SRMs once per 

day.

18 Lab replicate OMRL 1d Sample suffix “d” 

indicates lab replicate.

19 Lab blank blMMDDYYd —

20 Lab blank blMMDDYYe A minimum of five blanks 

should be run per day.

Continue analyzing samples, repeating above sequence from rows 

6–20 (do not repeat the SRMs). Always complete the run with a 

replicate followed by two blanks to ensure quality assurance.
Analytical Method 13

Analysis Procedure

General operational procedures and instrumental 

parameters essential to the proper execution of the method for 

analyzing absorbance and fluorescence on the Aqualog® are 

described here. A detailed description of the CAWSC OMRL 

standard operating procedure (SOP) is provided in appendix 1.

Initial preparations for sample analysis include warming 

up the instrument for 1 hour prior to the first scan. Lamp hours 

are recorded when the instrument is switched on because the 

lamp degrades over time, and the manufacturer recommends 

the lamp be replaced either at 1000 hours of use or after 1 year 

of installation. The OMRL staff replaces the lamp annually 

regardless of hours of lamp use. Always wear gloves when 

handling cuvettes and samples for the Aqualog®. All blanks, 

dilutions, and standards should be prepared using Type I LRW.

A typical analytical run includes three unique 

experiments: (1) Raman, (2) baseline, and (3) analytical 

set. The purpose of these experiments, the reagents used, 

and the Aqualog® experiment parameters used for each are 

described in table 6. Aqualog® software enables the analyst to 

create experiment files that contain specific information for 

the instrument parameters, including the experiment set-up 

and acquisition type (Horiba Scientific, 2011, page 13-4). 

Software setup should include creating these files for each of 

the three unique experiments (that is, water Raman, baseline, 

and analytical set) to improve run-time efficiency and to 

avoid inconsistent sample analysis. Further details regarding 

the Raman, baseline, and analytical set are given in the 

following paragraphs.

1. Raman scan. It should be emphasized that long-term 

stability of the instrument is one of the chief concerns 

of the manufacturer and the user, and one of the best 

known methods to achieve this is by performing the 

daily Raman scan using LRW. Routine examination of 

the water Raman spectrum serves as an early indicator of 

the instrument’s integrity. The Raman peak area is used 

to normalize fluorescence signals, thus producing data 

in units that can be compared across instruments. This 

scan should be performed at the start of each analytical 

run using LRW to verify that the peak location of the 

water Raman is 397 plus or minus 1 nm (fig. 7). The 

Raman area is calculated using the baseline-corrected 

peak boundary definition (Murphy and others, 2011) 

outside of the Aqualog® user interface and is determined 

after collecting a baseline scan (see experiment 2) using 

a processing routine developed by the OMRL staff. In 

the OMRL, the Raman area is expected to be within 

75 percent of the value obtained after the last lamp 

change. If the Raman peak location and area do not meet 

acceptance criteria, allow the instrument to warm up for 

another 30 minutes and repeat the scan. If acceptance 

criteria continue not to be met, examine common sources 

of interference or contamination (for example, cuvette 

blemishes, LRW contamination, lamp alignment) before 

contacting the manufacturer for technical support.

Table 6. Aqualog® experiments used in a typical analytical run, along with method, parameters, and reagents.

[CCD, charge-coupled device; Em, emission; Ex, excitation; LRW, lab reagent water; NY, New York; SRM, standard reference material; 3D, three-dimensional; 

=, equal to]

Scan type Aqualog® method Experiment parameters Purpose Reagent

Raman Spectra. Two-dimensional 

emission spectra

Integration time=10 

Accumulation=1 

Ex=350 

Em=1.64 (should be equivalent to 3D scan) 

CCD gain=medium 

Sample only 

Cuvette position=1

Used to normalize 

baseline, blanks and 

samples to the daily 

LRW.

Type 1 LRW

Baseline 3D. Three-dimensional 

emission spectra plus 

absorbance.

Integration time=1

Ex=600-240-3

Em=1.64

CCD gain=medium

Blank/sample setup=blank only

Cuvette position=1

Establishes a daily 

baseline for the 

instrument and is 

used to baseline 

correct scans 

(blanks, samples, and 

standards).

Type 1 LRW

Analytical 

set

3D. Three-dimensional 

emission spectra plus 

absorbance.

Integration time=1

Ex=600-240-3

Em=1.64

CCD gain=medium

Blank/sample setup=blank from file

Cuvette position=1

The analytical set 

includes samples, 

verification samples 

(SRMTea or knowns), 

sample duplicates, 

and laboratory blanks.

Blank reagent is organicfree Type 1 LRW. 

Verification sample 

(SRMTea) reagent 

is Pure Leaf ®, 

unsweetened black tea, 

Purchase, N.Y.
14 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

2. Baseline scan. The baseline scan must be collected prior 

to the analysis of samples to obtain the signal associated 

with the instrument, cuvette, and LRW in the absence of 

fluorescent DOM. The daily baseline scan is subtracted 

from the analytical set collected afterward (see next 

item). The baseline scan must be quality checked to 

determine whether it meets laboratory objectives. 

Quality check the baseline by confirming the response 

is less than the LT-MDL in the regions of interest. The 

OMRL staff has developed a processing routine that 

Raman-normalizes the baseline scan, calculates water 

Raman area and peak location, and generates three 

graphs for visual determination of baseline quality 

(fig. 8). The first graph shows the current baseline scan, 

the second graph shows the average of the long-term 

baseline subtracted from the daily baseline, and the 

third graph shows the difference between the standard 

deviation of the long-term average baseline and the daily 

baseline. If the baseline does not pass quality-assurance 

standards (for example, it exceeds the LT-MDL), allow 

the instrument to warm up for another 30 minutes and 

repeat along with the water Raman scan. If acceptance 

criteria continue not to be met, examine common sources 

of interference or contamination (for example, cuvette 

blemishes, LRW contamination) before contacting the 

manufacturer for technical support.

3. Analytical set. Only after the water Raman and baseline 

scans have met laboratory-defined objectives should the 

analytical set begin. An analytical set includes samples, 

verification samples (that is, SRMTea or knowns), 

laboratory replicates, and laboratory blanks. A typical 

analytical set scan order is presented in table 5.

Examination of QA/QC in the analytical set should be 

done to determine sample reruns and dilution requirements. 

Fluorescence measurements of highly concentrated samples 

are subject to measurement errors like detector saturation and 

inner-filtering effects (IFE). Previous studies indicate the IFE 

is linear and correctable for most natural samples when A254

is between 0.03–0.3 absorbance units (AU) when measured 

in a 1 centimeter (cm) cuvette (for example, Ohno, 2002; 

Lakowicz, 2006, Miller and others, 2010). Most undiluted 

natural samples from riverine, lake, and estuarine systems far 

exceed that level, however, as do samples associated with high 

concentrations of DOM inputs, such as those of wastewater 

effluent or with oil and gas contamination. To standardize the 

correction procedure, all samples initially are analyzed at full 

concentration, and the A255 value is noted. If the A255 exceeds 

0.3 AU, the sample is diluted with LRW to a concentration 

at which the A255 is in the range of 0.03 to 0.3 AU. Note that 

use of A255 reflects the output of the Aqualog® method for 

simultaneous collection of fluorescence and absorbance data, 

where absorbance data are collected in 3-nm increments from 

240 to 600 nm.

It should be emphasized that this value of 0.3 AU at 

A255 was developed for samples containing a “typical” 

array of DOM compounds (fig. 6); however, samples that 

have particularly high absorbance and fluorescence in other 

regions of EEM space may require further dilution to avoid 

interferences due to IFE or instrument detector saturation in 

those regions. For example, a sample containing very high 

fluorescence at low-UV wavelengths could require additional 

dilution so that the absorbance and fluorescence response 

falls within a linear range of the instrument across the entire 

EEMs space.

Data Reporting and Limits

Given there is not a CRM for optical measurements from 

which to calculate a minimum detection limit (MDL) across 

the absorbance spectrum or EEM space, the OMRL staff 

has developed reporting limits by modifying the approach 

recommended by Childress and others (1999) and the 

U.S. Environmental Protection Agency (2000). The LT-MDL 

is reported as three times the standard deviation plus the 

average of baseline-corrected blanks collected over a 2-year 

period. Three times the standard deviation is a commonly 

used estimate of the measured response corresponding to the 

critical Student’s t value at a significance level of 99 percent, 

as referenced in Childress and others (1999).

Absorbance LT-MDL was calculated across the spectrum 

(240–600 nm) from 1200 baseline-corrected instrument 

blanks analyzed over a 2-year period (January 2013–

December 2014). Absorbance is reported in absorbance units 

(AU) obtained directly from the instrument. Absorbance 

LT-MDLs vary by wavelength, ranging from 0.01 AU at A240 to 

0.004 AU at A600 (fig. 9).

Fluorescence LT-MDL was calculated at each ex-em 

pair determined from 1200 baseline-corrected, water Ramannormalized blanks collected during the same 2-year period 

as absorbance (January 2013–December 2014). Fluorescence 

data are expressed in Raman-normalized intensity units (RU; 

Murphy and others, 2010). Fluorescence LT-MDLs vary by 

excitation-emission pairs, ranging from 0.004 RU throughout 

much of the EEM spectra to 0.1 RU in the region of peak B 

(ex275, em304; fig. 10).
Analytical Method 15

sac17-0642_fig07ab

1,000

1,500

500

0

2,000

2,500

3,000

375 380 385 390 395 400 405 410 415 420 425

Signal height, in arbitrary units

Emission wavelength, in nanometers

A

B

Peak position at emission 397 nanometers

2,000

2,100

2,200

2,300

2,400

2,500

2,600

2,700

2,800

2,900

3,000

Water Raman area, in arbitrary units

EXPLANATION

Raman area

Lamp change

Dec. 7, 2012

Mar. 17, 2013

June 25, 2013

Oct. 3, 2013

Jan. 11, 2014

April 21, 2014

July 30, 2014

Nov. 7, 2014

Feb. 15, 2015

May 26, 2015

Sept. 3, 2015

Dec. 12, 2015

Mar. 21, 2016

June 29, 2016

Oct. 7, 2016

Figure 7. Aqualog® water Raman criteria: A, peak position should be 397 nanometers plus or minus 1 nanometer; and B, the area 

should be at least 75 percent of the value obtained after the last lamp change.
16 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

sac17-0642_fig08

A

550

600

450

500

350

400

250

250 300 350 400 450 500 550

0.5

0.4

0.3

0.2

0.1

0

–0.1

–0.2

–0.4

–0.3

–0.5

600

300

Excitation, in nanometers

B

550

600

450

500

350

400

250

250 300 350 400 450 500 550

1.0

–0.5

0.5

0

600

300

Excitation, in nanometers

C

Emission, in nanometers Emission, in nanometers Emission, in nanometers

550

600

450

500

350

400

250

250 300 350 400 450 500 550

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

600

300

Excitation, in nanometers

Figure 8. Visual inspection of the daily baseline excitationemission matrix (EEM): A, the daily baseline; B, the average of the 

long-term average baseline subtracted from the daily baseline; 

and C, the standard deviation of the long-term average baseline 

subtracted from the daily baseline. The EEM plots should be free 

of a response in area of interest.

Acceptance of Data

Optical properties are measured within quality-control 

thresholds, as indicated by laboratory standards, which for 

the OMRL include monthly measurements of potassium 

dichromate and quinine sulfate, daily measurement of a 

standard reference material (Pure Leaf ®, unsweetened black 

tea, Purchase, New York), and laboratory replicates measured 

approximately every 10 samples. Results for analytical sets 

are considered acceptable if all standards are within 20 percent 

of the target value, laboratory replicates are within 5 percent 

RPD, and laboratory blanks (that is, LRW) are less than onetenth of the LT-MDL. Analytical runs are evaluated daily by 

the analyst to ensure all data-quality indicators meet laboratory 

objectives and are reviewed by the quality-assurance manager. 

If standards, replicates, and blanks are not within acceptable 

thresholds, all analyses in that set must be repeated. Because 

the recommended sample hold time for optical analysis is 

2 days following sample collection, sample reruns should be 

done as soon as possible.
Analytical Method 17

sac17-0642_fig09 Intensity, in absorbance units

Wavelength, in nanometers

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

0.011

Figure 9. Absorbance spectra measured between 240 and 600 nanometers indicating the long-term method detection limit calculated 

using 1200 baseline-corrected blanks analyzed during January 2013–December 2014.

280

300

320

340

360

380

400

420

440

460

480

500

520

540

560

580

600

240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

Excitation, in nanometers

Emission, in nanometers

EXPLANATION

Intensity, in Raman units

0 to 0.15

>0.15

Figure 10. Fluorescence excitation-emission matrix indicating the long-term method detection limit calculated using 1200 baselinecorrected blanks analyzed January 2013–December 2014.
18 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

Data Processing and Corrections

Data processing is performed using processing routines 

developed by the OMRL staff. These processing routines 

are laboratory specific because they are dependent on the 

sample- and analytical batch-naming conventions and datastorage structure established by the OMRL staff, as well as on 

the instrument setup and lab-defined QA/QC measurements. 

A description of the processing and correction of absorbance 

and fluorescence measurements is given here.

Data organization is paramount for effective processing, 

retrieval, compilation, and analyses; thus, establishing a 

consistent file-storage system and standardized sample-naming 

convention is highly recommended. For reference, an example 

showing the OMRL folders and files created from one 

analytical run date (May 18, 2016) is shown in table 7.

All data produced by the Aqualog® using the analysis 

procedure in this report are instrument- and baselinecorrected. The instrument correction factors account for 

wavelength-dependent components of the system, like the 

xenon lamp, gratings, and signal detectors. A set of instrument 

correction factors—instrument specific and provided by 

the manufacturer—for excitation (that is, xcorrect.spc) and 

emission (that is, mcorrect.spc) are automatically applied by 

the software to each scan (Horiba Scientific, 2011, page 13-2). 

Baseline correction is applied by the software during analysis, 

as described previously.

All other data corrections are completed in MATLAB 

v8.5 R2015a. Performing remaining data treatment outside of 

the Aqualog® software environment ensures standardization 

of data and better comparability among datasets and 

across instruments.

Upon completion of an analytical run, baseline-corrected 

and instrument-corrected data are exported to the desired 

storage location using the Aqualog® HJY batch export feature 

(appendix 1). A MATLAB data processing script developed by 

OMRL staff then is used to perform corrections (for example, 

water-Raman normalization, correct for IFE), calculations 

(for example, multiply diluted samples to the appropriate 

concentration, calculate designated slopes and indices), 

data manipulation (for example, Rayleigh trims, vectorize 

fluorescence data), compile summary data (for example, 

extract a subset of widely published absorbance wavelengths, 

fluorescence ex-em pairs, and indices), evaluate QA data, 

assign data qualifiers (for example, processed summary 

reports for absorbance and fluorescence; appendixes 4 and 

5, respectively), and manage data structure (for example, 

add samples to MATLAB structure array where related data 

types are stored in fields). The OMRL structure array stores 

complete absorbance spectra and full excitation-emission 

matrices for all samples analyzed in the laboratory, including 

all QA/QC samples (for example, daily laboratory blanks, 

replicates, and SRMTea and monthly Starna potassium 

dichromate and reference-cell scans). Storage in this type of 

array allows for easy data retrieval for modeling and reporting.

Data Storage

The Aqualog® collects high-resolution scans that include 

thousands of individual datum points for each sample; thus, 

data storage is not trivial. The OMRL staff archives the rawdata files and processed-data files on an internal network 

server. These files are available upon request and include the 

Aqualog® software project file; original exported data files 

including baseline, water Raman, sample and blank scans; and 

processed files containing full-absorbance scans, fluorescence 

EEMs, and laboratory QA data.

Only a subset of these data from selected projects and 

samples for which sites have been established are available 

to the public through the USGS National Water Information 

System (NWIS); these are limited to the commonly published 

diagnostic parameters and indices listed in table 8. The 

diagnostic wavelengths and EEMs pairs are transferred to 

the OMRL internal database when all QA/QC have been 

evaluated and accepted.
Data Storage 19

Aqualog - DOM

– Aqua DOM 2016

– q2016-05-18

– Exported Aqualog Files

– qa2016-05-18

– bl051816a.csv

bl051816b.csv

bl051816c.csv

bl051816d.csv

bl051816e.csv

gr20329.csv

gr20329d.csv

gr20330.csv

gr20331.csv

gr20332.csv

gr20332s50.csv

gr20334.csv

gr20335.csv

gr20335s50.csv

gr20336.csv

gr20336s10.csv

gr20337.csv

gr20338.csv

gr20339.csv

grSRMHTs01.csv

grSRMHUs01.csv

Aqualog - DOM

– Aqua DOM 2016—Continued

– q2016-05-18—Continued

– Exported Aqualog Files—Continued

– qf2016-05-18

bl051816a.dat

bl051816b.dat

bl051816c.dat

bl051816d.dat

bl051816e.dat

gr20329.dat

gr20329d.dat

gr20330.dat

gr20331.dat

gr20332.dat

gr20332s50.dat

gr20334.dat

gr20335.dat

gr20335s50.dat

gr20336.dat

gr20336s10.dat

gr20337.dat

gr20338.dat

gr20339.dat

grSRMHTs01.dat

grSRMHUs01.dat

wr051816.dat

Table 7. The Organic Matter Research Laboratory data-storage structure for example analysis date of May 18, 2016.

[Files are in alpha-numeric order. Sample suffix “s” followed by numbers denotes diluted sample percent concentration. Sample suffix “d” indicates a 

laboratory replicate scan. File extension .csv indicates a comma-separated file and .dat indicates a generic data file created upon export by Aqualog software. 

Abbreviation: DOM, dissolved organic matter]
20 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

Table 8. National Water Information System (NWIS) codes for commonly published diagnostic parameters and indices.

[cm, centimeter; em, emission; ex, excitation; FDOM, fluorescent dissolved organic matter; nm, nanometer; RU, Raman units; UV, ultraviolet; wf, filtered water]

Method tool Analyte Result name Parameter definition Method 

code

Parameter 

code

Horiba Scientific 

Aqualog®

Absorbance A254 Absorbance, 254 nm, water, filtered, absorbance units 

per centimeter.

UV008 50624

Horiba Scientific 

Aqualog®

Absorbance SUVA254 Specific UV absorbance, 254 nm, water, filtered, 

1-cm path length, calculated, liter per milligram of 

dissolved organic carbon per meter.

UV003 63162

Horiba Scientific 

Aqualog®

Absorbance A280 Absorbance at 280 nm, water, filtered, absorbance 

units per centimeter.

ABS01 32296

Horiba Scientific 

Aqualog®

Absorbance A370 Absorbance at 370 nm, water, filtered, absorbance 

units per centimeter.

ABS01 32297

Horiba Scientific 

Aqualog®

Absorbance A412 Absorbance at 412 nm, water, filtered, absorbance 

units per centimeter.

ABS01 32298

Horiba Scientific 

Aqualog®

Absorbance A440 Absorbance at 440 nm, water, filtered, absorbance 

units per centimeter.

ABS01 32299

Horiba Scientific 

Aqualog®

Absorbance S275–295 Absorption spectral slope, wavelengths 275–295 nm, 

unitless.

ABS02 32300

Horiba Scientific 

Aqualog®

Absorbance S290–350 Absorption spectral slope, wavelengths 290–350 nm, 

unitless.

ABS02 32301

Horiba Scientific 

Aqualog®

Absorbance S350–400 Absorption spectral slope, wavelengths 350–400 nm, 

unitless.

ABS02 32302

Horiba Scientific 

Aqualog®

Absorbance S412–600 Absorption spectral slope, wavelengths 412–600 nm, 

unitless.

ABS02 32331

Horiba Scientific 

Aqualog®

Fluorescence Peak A Fluorescence, wf, ex260 em450 RU. FL020 32304

Horiba Scientific 

Aqualog®

Fluorescence Peak B Fluorescence, wf, ex275 em304 RU. FL020 32305

Horiba Scientific 

Aqualog®

Fluorescence Peak C Fluorescence, wf, ex340 em440 RU. FL020 52901

Horiba Scientific 

Aqualog®

Fluorescence Peak D Fluorescence, wf, ex390 em510 RU. FL020 32307

Horiba Scientific 

Aqualog®

Fluorescence FDOM Fluorescence, wf, ex370 em460 RU. FL020 52902

Horiba Scientific 

Aqualog®

Fluorescence Peak M Fluorescence, wf, ex300 em390 RU. FL020 32309

Horiba Scientific 

Aqualog®

Fluorescence Peak N Fluorescence, wf, ex280 em370 RU. FL020 32310

Horiba Scientific 

Aqualog®

Fluorescence Peak T Fluorescence, wf, ex275 em340 RU. FL020 32311

Horiba Scientific 

Aqualog®

Fluorescence FI Fluorescence index—ratio of emission intensities that 

characterizes the slope of the emission curve at an 

excitation of 370 nm.

FL020 32312

Horiba Scientific 

Aqualog®

Fluorescence HIX Humification index—calculated by dividing the 

fluorescence intensity in the 435–480 nm region by 

the total intensities in the 300–345 and 435–480 nm 

regions.

FL020 32313
Data Analysis 21

Data Analysis

The approach to optical data analysis depends on the 

objective of the study and the scale of measurement (that is, 

quantitative or qualitative). As mentioned before and described 

in table 1, discrete diagnostic wavelengths and indices can 

be extracted from the absorbance and fluorescence scans. 

In addition, multi-parameter techniques can be used that 

incorporate all the data (that is, complete absorbance spectra 

and fluorescence matrices). A brief overview of some of the 

ways optical measurements of DOM have been used, with 

a focus on approaches used by the OMRL staff, is provided 

here. For additional information, the reader is referred to 

an extensive body of literature on this topic (Coble, 1996; 

McKnight and others, 2001; Stedmon and others, 2003; 

Fellman and others, 2010; Coble and others, 2014).

The measurement of ultraviolet absorbance has been 

used by the drinking-water industry for decades and has been 

shown to be a useful proxy for DOC concentration (that is, 

A254; Edzwald and others, 1985; Rathbun, 1996; Korshin and 

others, 1997; Sadiq and Rodriguez, 2004) and disinfection 

byproduct (DBP) precursor concentrations (that is, A272; 

Korshin and others, 2002; Li and others, 2002; Roccaro and 

others, 2008). In addition to concentration, absorbance data 

can provide insight into the chemical composition of the DOM 

pool. Carbon normalization of the intensity of the response at 

a given absorbance wavelength (that is, SUVA254 calculated as 

A254 value divided by DOC concentration, reported in L/mg 

C/m) has been shown to be correlated with the hydrophobic 

organic acid fraction of DOM (Spencer and others, 2012) 

and is a useful proxy for DOM aromatic content (Weishaar 

and others, 2003) and molecular weight (Chowdhury, 2013). 

Similarly, spectral slopes and slope ratios have been related to 

the relative molecular weight and aromaticity of DOM (Chin 

and others, 1994; Helms and others, 2008); lower values are 

generally indicative of higher molecular weight DOM, which 

can change upon photolytic exposure (Helms and others, 

2008; Spencer and others, 2009; Hansen and others, 2016).

Fluorescence intensity at a given ex-em pair also can be 

related to DOC concentration, DBP precursor concentrations 

(Kraus and others, 2010; Beggs and Summers, 2011; 

Carpenter and others, 2013), biological oxygen demand 

(Baker and Curry, 2004; Hudson and others, 2008), mercury 

concentrations (Fleck and others, 2014), and wastewater 

content (Goldman and others, 2012). Like absorbance data, 

fluorescence data also are widely used to gain insight into 

DOM composition, source, and reactivity. Information about 

DOM composition can be inferred from the presence or 

absence of fluorophores, shifts in peak maxima, ratios of 

peaks, and calculated indices, as well as by normalizing each 

ex-em pair by DOC concentration (Beggs and Summers, 

2011). Commonly reported DOM compositional indicators 

include the fluorescence index, humification index, freshness 

index, and biological index (table 1).

Although these diagnostic wavelengths and indices can 

be very informative, they often miss information that can be 

gleaned by using the full absorbance and fluorescence dataset 

generated by instruments like the Aqualog®. Other approaches 

to data interpretation involve the use of multivariate and multiway modeling techniques (Stedmon and others, 2003; Fellman 

and others, 2008). In the following paragraphs, we discuss 

three commonly used approaches—principal component 

analysis (PCA), discriminant analysis (DA), and parallel 

factor analysis (PARAFAC)—and then provide more detailed 

information about tools the OMRL staff have developed to 

run PARAFAC.

Principal component analysis (PCA) is one of the 

most frequently used multivariate techniques to explore 

optical data (for example, Stepanauskas and others, 2003; 

Baker and others, 2008; Jaffé and others, 2008; Kraus and 

others, 2008; Dalzell and others, 2009; Miller and McKnight, 

2010; Goldman and others, 2012, 2014). The primary aim 

of PCA is dimension reduction, in which uncorrelated linear 

combinations called principal components are identified. The 

first component explains the most variability in a dataset, 

the second component explains the second-most, and so on. 

Model output displays scores (clustering and/or separation of 

objects and their relationship to the principal components) and 

loadings (the original loadings projected onto the principal 

axes; fig. 11).

Discriminant analysis (DA) is a predictive method that 

classifies a sample to a user-designated group on the basis 

of known responses. Although DA is performed more rarely 

than PCA, it has potential to improve our understanding of 

DOM biogeochemical processes because of its ability to 

analyze complex spatial relationships, and it allows for the 

identification of the most important discriminating variables 

that lead to proper assignment of a sample to a designated 

group (for example, Spencer and others, 2007; Hansen and 

others, 2016; fig. 12).

Parallel factor analysis (PARAFAC), a multi-way 

statistical approach for detecting underlying structures in 

complex datasets like fluorescence EEMs, has proven to be 

a powerful tool for studying DOM (Bro, 1997) and has been 

widely applied in combination with other measurements to 

rapidly quantify and characterize DOM across a range of 

environments (for example, Stedmon and Markager, 2005; 

Fellman and others, 2008; Kowalczuk and others, 2009). The 

aim of PARAFAC is to distill fluorescence signatures into 

distinct fluorescence components (fluorophores) that are not 

restricted to a single excitation-emission pair.

Unlike PCA or DA, which are common statistical 

methods available in many software packages (for instance, 

OMRL uses SAS JMP v.12.2) PARAFAC requires the use 

of MATLAB, or a similar high-performance programming 

language (for example, Python or R), that allows users to 

perform matrix calculations, develop and run algorithms, and 

create data visualization products and user interfaces.

In order to run PARAFAC in MATLAB, the data 

structure must be arranged in a specific format according to 

the procedures in Stedmon and Bro (2008).
22 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

PARAFAC model validation is determined by examining 

a variety of methods in combination (for example, analysis 

of spectral properties, split-half analysis, residual analysis, 

and random initialization). Although there is no quantitative 

measure of “best fit” (Murphy and others, 2013), subjectivity 

in determining the final modeled components can be 

minimized by evaluating each dataset carefully using the 

diverse validation methods explained by Stedmon and 

Bro (2008).

When validated, component loadings are interpreted 

to represent individual fluorophores and can be examined 

by the absolute value (that is, component loadings), which 

is a reflection of the intensity of fluorescence, and by the 

relative contribution to total fluorescence (that is, component 

percentage loadings), which is a reflection of composition 

(fig. 13). Model component loadings can be used for further 

analysis of the individual observations like “new parameters” 

in other multivariate approaches discussed previously, such 

as PCA (for example, Kraus and others, 2008; Miller and 

McKnight 2010; Fleck and others, 2014) or DA (Spencer and 

others, 2007; Hansen and others, 2016), to gain insight into 

DOM composition.

sac17-0642_fig11

A B

–4

–3

–2

–1

0

1

2

3

4

Component 2 (17.1%)

–4 –3 –2 –1 0 1 2 3 4

Component 1 (40.9%)

–1.0

–0.5

0.0

0.5

1.0

Component 2 (17.1%)

S350–400

–1.0 –0.5 0.0 0.5 1.0

Component 1 (40.9%)

C:M

SVA555

C:A S275–295

FI

β:α

EXPLANATION

Study unit A

Study unit B

Study unit C

Study unit D

Study unit E

Figure 11. Example of model output from principal component analysis: A, the score plot displays clustering or separation of individual 

samples and their relationship to the principal components; and B, the loadings plot graphs the original loadings projected onto the 

principal axes. (Original loadings correspond to selected optical measurements, labels abbreviated as in table 1.)
Data Analysis 23

sac17-0642_fig12

SVA555

S275–295

S350–400

FI

C:A

C:M

β:α

Canonical 1

EXPLANATION

Study unit A Abbreviations

Study unit B

Study unit C

Study unit D

Study unit E

S

C:A

SVA

C:M

Fl

Slope

Fluorescence ratio of Peak to Peak A

Specific visible absorbance

Fluorescence ratio of Peak to Peak M

Fluorescence

–8

–6

–4

–2

0

2

4

6

8

Canonical 2

–8 –6 –4 –2 0 2 4 6 8 Study unit D Study unit A Study unit C Study unit B Study unit E

Figure 12. Example of model output from discriminant analysis. The canonical plot shows the two dimensions (canonicals 1 and 2) 

that provide the best separation among user-defined groups. Points are individual samples; inner ellipses represent the 95-percent 

confidence interval for group means and outer ellipses represent the region estimated to contain 50 percent of the population of each 

group; and the biplot ray represents the covariates and the degree of association of the covariates with the two canonical variables.
24 Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from DOM

sac17-0642_fig13

A

B

CIntensity, in Raman units Composition, in percent

Sample number

0

0 50 100 150 200 250

20

40

60

80

100

0

0.1

0.2

0.3

0.4

0.5

Component 1

250 300 350

Excitation, in nanometers

Emission, in nanometers

400 450

550

450

350

300

400

600

500

Component 2 Component 3 Component 4 Component 5

250 300 350

Excitation, in nanometers

400 450

550

450

350

300

400

600

500

250 300 350

Excitation, in nanometers

400 450

550

450

350

300

400

600

500

250 300 350

Excitation, in nanometers

400 450

550

450

350

300

400

600

500

250 300 350

Excitation, in nanometers

400 450

550

450

350

300

400

600

500

EXPLANATION

Model loadings

Component 1

Component 2

Component 3

Component 4

Component 5

EXPLANATION

Model loadings

Component 1

Component 2

Component 3

Component 4

Component 5

0.22

0.18

0.14

0.01

0.06

0.02

Intensity, in Raman units

Figure 13. Example of model output from parallel factor analysis (PARAFAC): A, component loadings are interpreted to represent 

individual fluorophores and can be examined; B, by absolute value, which is a reflection of the intensity of fluorescence; and C, by the 

relative contribution to total fluorescence, which is a reflection of composition.
References Cited 25

Summary

The purpose of this document is to provide a standardized 

method for the collection, correction, and interpretation of 

optical data produced by the Aqualog®. Optical measurements 

such as absorbance and fluorescence are easy to take; however, 

they can be affected by many variables rendering them less 

comparable, including such things as inconsistencies in 

sample collection (for example, filter pore size, preservation), 

the application of corrections for interferences (for example, 

inner-filtering corrections), differences in holding times, and 

instrument drift (for example, lamp intensity). A documented, 

standardized procedure to address these variables ensures 

that the optical (absorbance and fluorescence) measurements 

collected by U.S. Geological Survey (USGS) researchers are 

useful and widely comparable.

Rigorous and quantifiable quality assurance and quality 

control are essential for making these data comparable, 

particularly because there is no published guideline for the 

measurement of dissolved organic matter (DOM) absorbance 

and fluorescence, and especially because there is no National 

Institute of Standards and Technology standard for DOM. 

Validation and quality-control samples are analyzed on 

a monthly basis to determine laboratory and instrument 

precision and daily (that is, each day samples are run) to 

ensure repeatability. Data are not considered acceptable unless 

they meet laboratory criteria: All standards should be within 

20 percent of the target value, laboratory replicates should be 

within 5 percent relative percent difference, and laboratory 

blanks (that is, laboratory reagent-grade water) should be less 

than one-tenth of the long-term method detection limit.

Finally, for data to be useful, they must be accessible to 

users in a format that can be easily analyzed and interpreted. 

The Organic Matter Research Laboratory staff has developed 

a processing routine that extracts a subset of the data, which 

is made available to the public through the USGS National 

Water Quality Information System (http://nwis.waterdata.usgs.

gov/usa/nwis/qwdata), and organizes the full datasets (that 

is, complete absorbance spectra and fluorescence excitationemission matrices) in different forms that allow for these data 

to be analyzed using multi-parameter and multi-way statistical 

approaches.

Acknowledgments

We thank Erica DeParsia, Amaryl Griggs, 

Quratulain Ahmed, and Sydney Berrios for laboratory 

assistance and logistical support. We appreciate reviews 

provided by Peter Lenaker and Emily Woodward which 

improved the quality and presentation of this work. Funding 

was provided by the California Water Science Center, 

Sacramento, California.

References Cited

Baker, A., and Curry, M.P., 2004, Fluorescence of 

leachates from three contrasting landfills: Water 

Research, v. 38, no. 10, p. 2605–2613, 

https://doi.org/10.1016/j.watres.2004.02.027.

Baker, A., and Spencer, R.G.M., 2004, Characterization 

of dissolved organic matter from source to sea using 

fluorescence and absorbance spectroscopy: Science of the 

Total Environment, v. 333, no. 1–3, p. 217–232, 

https://doi.org/10.1016/j.scitotenv.2004.04.013.

Baker, A., Elliott, S., and Lead, J.R., 2007, Effects of 

filtration and pH perturbation on freshwater organic matter 

fluorescence: Chemosphere, v. 67, no. 10, p. 2035–2043, 

https://doi.org/10.1016/j.chemosphere.2006.11.024.

Baker, A., Bolton, L., Newson, M.D., and Spencer, R.G.M., 

2008, Spectrophotometric properties of surface water 

dissolved organic matter in an afforested upland peat 

catchment: Hydrological Processes, v. 22, no. 13, p. 2325–

2336, https://doi.org/10.1002/hyp.6827.

Beggs, K.M.H., and Summers, R.S., 2011, Character and 

chlorine reactivity of dissolved organic matter from a 

mountain pine beetle impacted watershed: Environmental 

Science and Technology, v. 45, no. 13, p. 5717–5724, 

https://doi.org/10.1021/es1042436.

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Appendixes 31

Appendix 1. Aqualog® Standard Operating Procedure Walkthrough

The Aqualog® Standard Operating Procedure (SOP) Walkthrough provides detailed operational procedures and instrumental 

parameters essential to the proper execution of the method for using the Horiba Scientific Aqualog® fluorometer to measure 

absorbance and fluorescence from dissolved organic matter. Click here to view.

Appendix 2. Processed Summary Report for Absorbance Data

A processed summary report for absorbance data is created by a MATLAB processing routine developed by the Organic 

Matter Research Laboratory staff. This summary report is intended to provide users of the Aqualog® fluorometer with a 

standardized approach by which to evaluate and validate data produced by the Aqualog® fluorometer. The processed summary 

report for absorbance data includes calculations (for example, multiply diluted samples to appropriate concentration, calculate 

designated slopes and indices), data manipulation (for example, compile summary data, extract a subset of widely published 

absorbance wavelengths and indices), evaluation of quality-assurance data, and assignment of data qualifiers. Click here to view.

Appendix 3. Processed Summary Report for Fluorescence Data

A processed summary report for fluorescence data is created by a MATLAB processing routine developed by the Organic 

Matter Research Laboratory staff. This summary report is intended to provide users of the Aqualog® fluorometer with a 

standardized approach by which to evaluate and validate data produced by the Aqualog® fluorometer. The processed summary 

report for fluorescence data includes corrections (for example, water Raman normalization, correct for inner-filtering effects), 

calculations (for example, multiply diluted samples to appropriate concentration, calculate designated slopes and indices), data 

manipulation (for example, Rayleigh trims, vectorize fluorescence data, compile summary data; for example, extract a subset of 

widely published fluorescence excitation-emission pairs, indices), evaluation of quality-assurance data, and assignment of data 

qualifiers. Click here to view.

Appendixes

Publishing support provided by the U.S. Geological Survey

 Science Publishing Network, Sacramento Publishing Service Center

For more information concerning the research of this report, contact the

 Director, California Water Science Center

 U.S. Geological Survey

 6000 J Street, Placer Hall

 Sacramento, California 95819

 http://ca.water.usgs.gov
Hansen and others—Procedures for Using the Horiba Scientific Aqualog® Fluorometer to Measure Absorbance and Fluorescence from Dissolved Organic Matter—

Open File Report 2018–1096

ISSN 2331-1258 (online)

https://doi.org/10.3133/ofr1096