Methods of Analysis by the U.S. Geological Survey 

National Water Quality Laboratory—Evaluation of Alkaline 

Persulfate Digestion as an Alternative to Kjeldahl Digestion 

for Determination of Total and Dissolved Nitrogen and 

Phosphorus in Water

______________________________________________________________________

Water-Resources Investigations Report 03–4174

U.S. Department of the Interior

U.S. Geological Survey
Methods of Analysis by the U.S. Geological Survey 

National Water Quality Laboratory—Evaluation of Alkaline 

Persulfate Digestion as an Alternative to Kjeldahl Digestion 

for Determination of Total and Dissolved Nitrogen and 

Phosphorus in Water

By Charles J. Patton and Jennifer R. Kryskalla

______________________________________________________________________

U.S. Geological Survey

Water-Resources Investigations Report 03–4174

Denver, Colorado

2003
U.S. DEPARTMENT OF THE INTERIOR

Gale A. Norton, Secretary

U.S. GEOLOGICAL SURVEY

Charles G. Groat, Director

The use of trade, product, or firm names in this report is for descriptive purposes only and does 

not imply endorsement by the U.S. Government.

_____________________________________________________________________________

For additional information write to: Copies of this report can be purchased from:

U.S. Geological Survey U.S. Geological Survey

Chief, National Water Quality Laboratory Branch of Information Services

Box 25046, Mail Stop 407 Box 25286

Federal Center Federal Center

Denver, CO 80225-0046 Denver, CO 80225-0286
Contents iii

CONTENTS

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

Introduction .......................................................................................................................................................... 2

Purpose and scope .......................................................................................................................................... 3

Acknowledgments.......................................................................................................................................... 3

Analytical method ............................................................................................................................................... 4

1. Application .............................................................................................................................................. 4

2. Method summary and considerations...................................................................................................... 4

3. Interferences ............................................................................................................................................ 5

4. Instrumentation and auxiliary analyses ................................................................................................... 8

5. Apparatus................................................................................................................................................. 9

6. Reagents .................................................................................................................................................. 10

7. Calibrants and quality-control solutions.................................................................................................. 12

8. Sample preparation.................................................................................................................................. 14

9. Instrument performance........................................................................................................................... 15

10. Calibration ............................................................................................................................................... 15

11. Procedure and data evaluation................................................................................................................. 15

12. Calculations ............................................................................................................................................. 17

13. Reporting results...................................................................................................................................... 18

14. Detection levels, bias, and precision ....................................................................................................... 18

Analytical performance and comparative results................................................................................................. 20

Summary and conclusions.................................................................................................................................... 26

References cited ................................................................................................................................................... 32

FIGURES

1. Analytical cartridge diagram for the air-segmented continuous flow analyzer (Alpkem RFA-300)

used to automate photometric determination of nitrate + nitrite in alkaline persulfate digests with a 

cadmium-reduction, Griess reaction method ........................................................................................... 6

2. Analytical cartridge diagram for the air-segmented continuous flow analyzer (Alpkem RFA-300)

used to automate photometric determination of orthophosphate in alkaline persulfate digests with a 

reduced phosphoantimonylmolybdenum blue reaction method .............................................................. 7

3. Typical calibration graph for total nitrogen determined as nitrate in alkaline persulfate digests ............ 16

4. Typical calibration graph for total phosphorus determined as orthophosphate in 

alkaline persulfate digests ........................................................................................................................ 16

5. Percent recovery of nitrogen from a series of 2.5 mg NH4

+-N solutions that contained increasing 

concentrations of organic carbon (OC) as glucose .................................................................................. 22

6. Histogram of postdigestion weight loss expressed as a percent of initial digest weight 

for one batch of 80 alkaline persulfate digests......................................................................................... 23

7. Logarithmic scatter plot of nitrate-corrected NAlkP concentrations (x-axis) and KN 

concentrations (y-axis) around the line of equal relation for 2,066 data pairs combined 

from large-scale validation experiments .................................................................................................. 24

8. Concentration differences between nitrate-corrected alkaline persulfate digestion nitrogen

(NAlkP) and Kjeldahl digestion nitrogen (KN) plotted about the line of zero difference as a 

function of nitrate concentration .............................................................................................................. 25
iv Contents

9. Boxplots of concentration differences between nitrate-corrected alkaline persulfate 

nitrogen (NAlkP) and Kjeldahl nitrogen (KN) for surface- and ground-water samples collected 

during nominally high- and low-flow condition ...................................................................................... 26

10. Logarithmic scatter plot of PAlkP concentrations (x-axis) and KP concentrations (y-axis) around 

the line of equal relation for 2,093 data pairs combined from large-scale validation experiments ......... 29

TABLES

1. Laboratory, parameter, and method codes for U.S. Geological Survey alkaline persulfate 

digestion total nitrogen and total phosphorus methods I-2650-03 and I-4650-03 ................................... 4

2. Data from a study of arsenate and arsenite interference in alkaline persulfate total 

phosphorus determinations....................................................................................................................... 8

3. Settings and operational details of Alpkem RFA-300 continuous flow analyzers used 

for this study............................................................................................................................................. 9

4. Volumes of mixed calibrant and amendment solution required to prepare working 

calibrants and blanks for determination of total nitrogen and phosphorus by the alkaline

persulfate digestion method...................................................................................................................... 12

5. Volumes of Environmental Resource Associates (ERA) DemandTM nutrient concentrate

used to prepare 1-liter volumes of check standards used in this study..................................................... 13

6. Suggested rack protocol for alkaline persulfate digest preparation ........................................................ 14

7. Suggested analyzer sample tray protocol for automated determination of nitrate and 

orthophosphate in alkaline persulfate digests........................................................................................... 17

8. Data and calculations used to estimate method detection limits (MDL) for nitrogen and

phosphorus in unacidified (FCC) and acidified (WCA) samples following alkaline 

persulfate digestion................................................................................................................................... 19

9. Most probable values and standard deviations for reference samples 9987L, 9987H, 

and 9987VH along with averages and standard deviations of these reference materials 

that were included in every rack of alkaline persulfate digests................................................................ 19

10. Spike recoveries of glycine and glycerophosphate in randomly selected high-flow and 

low-flow samples that were included in every rack of alkaline persulfate digests .................................. 20

11. Concentration differences between selected samples prepared and analyzed in duplicate in each

block of alkaline persulfate digests .......................................................................................................... 20

12. Recovery of inorganic and organic nitrogen from representative compounds......................................... 21

13. Recovery of organic phosphorus from representative compounds by alkaline persulfate and 

low-level acid persulfate digestion methods ............................................................................................ 21

14. Results of paired t-tests for nitrate-corrected alkaline persulfate dissolved nitrogen and

Kjeldahl dissolved nitrogen concentrations determined in filtered-water samples.................................. 27

15. Results of paired t-tests for nitrate-corrected alkaline persulfate total nitrogen and Kjeldahl

total nitrogen concentrations determined in acidified whole-water samples ........................................... 28

16. Results of paired t-tests for dissolved and total phosphorus concentrations determined in 

filtered and acidified whole-water samples by alkaline persulfate digestion (PAlkP) and Kjeldahl 

digestion methods..................................................................................................................................... 30
Contents v

CONVERSION FACTORS AND ABBREVIATED WATER-QUALITY UNITS

Degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) by using the following equation:

°F = (1.8 × °C) + 32.

ABBREVIATIONS AND ACRONYMS

A/D analog-to-digital converter

ASTM American Society for Testing and Materials

CCV continuing calibration verification

DI deionized water

DNAlkP alkaline persulfate dissolved nitrogen

DPAlkP alkaline persulfate dissolved phosphorus

FCA filtered, chilled, acidified (bottle type for USGS dissolved nutrient samples)

FCC filtered, chilled (bottle type for USGS dissolved nutrient samples)

FW formula weight

h hour

Hz hertz

i.d. inside diameter

KDN Kjeldahl dissolved nitrogen

KDP Kjeldahl dissolved phosphorus

KN used collectively for KDN and KTN

KTN Kjeldahl total nitrogen

KTP Kjeldahl total phosphorus

lb/in2 pounds per square inch

LRL laboratory reporting level

Multiply By To obtain

Length

centimeter (cm) 3.94 x 10-1 inch

micrometer (μm) 3.94 x 10-5 inch 

millimeter (mm) 3.94 x 10-2 inch

nanometer (nm) 3.94 x 10-8 inch

Volume

liter (L) 2.64 x 10-1 gallon 

liter (L) 33.81 ounce, fluid

microliter (μL) 2.64 x 10-7 gallon 

milliliter (mL) 2.64 x 10-4 gallon

Mass

gram (g) 3.53 x 10-2 ounce, avoirdupois

milligram (mg) 3.53 x 10-5 ounce, avoirdupois

Pressure

kilopascal (kPa) 1.45 x 10-1 pounds per square inch 
vi Contents

mg-As/L milligrams arsenic per liter

mg-C/L milligrams carbon per liter

mg-N/L milligrams nitrogen per liter

mg/L milligrams per liter

mg-P/L milligrams phosphorus per liter

M molarity (moles per liter)

max maximum

MDL method detection limit

MPV most probable value

N normality (equivalents per liter)

NAlkP used collectively for DNAlkP and TNAlkP

NED N-(1-Naphthyl)ethylenediamine dihydrochloride reagent

NOM natural organic matter

NWQL National Water Quality Laboratory

OC organic carbon

OWQ Office of Water Quality

o.d. outside diameter

PC personal computer

PBCdR packed-bed cadmium reactor

P/N part number

PTFE polytetrafluoroethylene

TNAlkP alkaline persulfate total nitrogen

TPAlkP alkaline persulfate total phosphorus

QC quality control

s second

sp. gr. specific gravity

SAN sulfanilamide reagent

SLS sodium lauryl sulfate

SOP standard operating procedure

SRWS U.S. Geological Survey Standard Reference Water Sample

STD CAL standard calibration control; adjusts absorbance range of photometric 

detectors used in this study

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey

v/v volume per volume

w/v weight per volume

WCA whole water, chilled, acidified (bottle type for USGS whole-water nutrient samples)

= equivalent to

> greater than

< less than

≤ less than or equal to

≈ nearly equal to

± plus or minus
Abstract 1

Methods of Analysis by the U.S. Geological Survey National 

Water Quality Laboratory—Evaluation of Alkaline Persulfate 

Digestion as an Alternative to Kjeldahl Digestion for 

Determination of Total and Dissolved Nitrogen and 

Phosphorus in Water

By Charles J. Patton and Jennifer R. Kryskalla

Abstract

Alkaline persulfate digestion was evaluated and 

validated as a more sensitive, accurate, and less toxic 

alternative to Kjeldahl digestion for routine 

determination of nitrogen and phosphorus in surfaceand ground-water samples in a large-scale and 

geographically diverse study conducted by U.S. 

Geological Survey (USGS) between October 1, 2001, 

and September 30, 2002. Data for this study were 

obtained from about 2,100 surface- and ground-water 

samples that were analyzed for Kjeldahl nitrogen and 

Kjeldahl phosphorus in the course of routine operations 

at the USGS National Water Quality Laboratory 

(NWQL). These samples were analyzed independently 

for total nitrogen and total phosphorus using an 

alkaline persulfate digestion method developed by the 

NWQL Methods Research and Development Program. 

About half of these samples were collected during 

nominally high-flow (April-June) conditions and the 

other half were collected during nominally low-flow 

(August-September) conditions. The number of 

filtered and whole-water samples analyzed from each 

flow regime was about equal.

By operational definition, Kjeldahl nitrogen 

(ammonium + organic nitrogen) and alkaline persulfate 

digestion total nitrogen (ammonium + nitrite + nitrate 

+ organic nitrogen) are not equivalent. It was 

necessary, therefore, to reconcile this operational 

difference by subtracting nitrate + nitrite concentrations from alkaline persulfate dissolved and total 

nitrogen concentrations prior to graphical and 

statistical comparisons with dissolved and total 

Kjeldahl nitrogen concentrations. On the basis of twopopulation paired t-test statistics, the means of all 

nitrate-corrected alkaline persulfate nitrogen and 

Kjeldahl nitrogen concentrations (2,066 paired results) 

were significantly different from zero at the p = 0.05 

level. Statistically, the means of Kjeldahl nitrogen 

concentrations were greater than those of nitratecorrected alkaline persulfate nitrogen concentrations. 

Experimental evidence strongly suggests, however, 

that this apparent low bias resulted from nitrate 

interference in the Kjeldahl digestion method rather 

than low nitrogen recovery by the alkaline persulfate 

digestion method. Typically, differences between 

means of Kjeldahl nitrogen and nitrate-corrected 

alkaline persulfate nitrogen in low-nitrate 

concentration (< 0.1 milligram nitrate nitrogen per 

liter) subsets of filtered surface- and ground-water 

samples were statistically equivalent to zero at the 

p = 0.05 level.

Paired analytical results for dissolved and total 

phosphorus in Kjeldahl and alkaline persulfate digests 

were directly comparable because both digestion 

methods convert all forms of phosphorus in water 

samples to orthophosphate. On the basis of twopopulation paired t-test statistics, the means of all 

Kjeldahl phosphorus and alkaline persulfate 

phosphorus concentrations (2,093 paired results) were 

not significantly different from zero at the p = 0.05 

level. For some subsets of these data, which were 

grouped according to water type and flow conditions at 

the time of sample collection, differences between 

means of Kjeldahl phosphorus and alkaline persulfate 

phosphorus concentrations were not equivalent to zero 
2 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

at the p = 0.05 level. Differences between means of 

these subsets, however, were less than the method 

detection limit for phosphorus (0.007 milligram 

phosphorus per liter) by the alkaline persulfate 

digestion method, and were therefore analytically 

insignificant.

This report provides details of the alkaline 

persulfate digestion procedure, interference studies, 

recovery of various nitrogen- and phosphoruscontaining compounds, and other analytical figures of 

merit. The automated air-segmented continuous flow 

methods developed to determine nitrate and 

orthophosphate in the alkaline persulfate digests also 

are described. About 125 microliters of digested 

sample are required to determine nitrogen and 

phosphorus in parallel at a rate of about 100 samples 

per hour with less than 1-percent sample interaction. 

Method detection limits for nitrogen and phosphorus 

are 0.015 milligram nitrogen per liter and 0.007 

milligram phosphorus per liter, respectively.

INTRODUCTION

Semiautomated, batch Kjeldahl digestion methods 

used at the U.S. Geological Survey (USGS) National 

Water Quality Laboratory (NWQL) for simultaneous 

nitrogen and phosphorus determinations in filteredand whole-water samples (Patton and Truitt, 1992, 

2000) are rapid and robust, but they suffer from several 

drawbacks, including:

• health and safety risks posed by concentrated 

acids, toxic reagents (mercury), and high 

temperatures (370°C);

• environmental effects and cost associated with 

processing and disposing of the mercurycontaining waste stream;

• propensity of acidic digests to trap and become 

contaminated by ammonia vapors in ambient 

laboratory air; and

• laboratory reporting limits (0.1 mg-N/L; 

0.04 mg-P/L) higher than those of other 

inorganic nitrogen- and phosphoruscontaining species, which limit the precision 

of mass balance estimates.

Alkaline persulfate digestion (Valderrama, 1981; 

Hosomi and Sudo, 1986; D’Elia and others, 1987; 

Ameel and others, 1993; D’Elia and others, 1997) 

provides a safer and more environmentally benign 

alternative to Kjeldahl digestion for routine, singledigest nitrogen and phosphorus determinations in 

water. Desirable characteristics of alkaline persulfate 

digestion compared to Kjeldahl digestion include:

• reagents that contain no mercury;

• fume hoods and acid scrubbers are not needed 

because digestion occurs in sealed tubes 

inside an autoclave;

• post-digestion contamination by ambient 

ammonia vapors is not a problem because all 

nitrogen-containing compounds are oxidized 

to and determined as nitrate;

• laboratory reporting limits (0.03 mg-N/L; 

0.01 mg-P/L) are similar to those of inorganic 

nitrogen- and phosphorus-containing 

nutrients; and

• waste-stream processing and disposal are 

straightforward.

During the past 15 years, alkaline persulfate 

digestion methods have been widely applied for 

estuarine and marine water analysis in preference to 

Kjeldahl digestion methods. Kjeldahl digestion 

methods continue to be widely applied for freshwater 

analysis, possibly because alkaline persulfate digestion 

methods are not approved for National Pollution 

Discharge Elimination System (NPDES) and Safe 

Drinking Water Act (SDWA) compliance monitoring. 

Nonetheless, an alkaline persulfate digestion method 

for total nitrogen determination (method 4500-N C, 

which does not include determination of phosphorus) is 

included in the 20th Edition of Standard Methods 

(American Public Health Association, 1998b, p. 4-102 

and 4-103). Note, however, that the method described 

in this report differs in two important respects from 

method 4500-N C. First, method 4500-N C states 

“samples preserved with acid cannot be analyzed [...].” 

The method described in this report is applicable to 

acidified nutrient samples—USGS FCA (filtered, 

chilled, acidified) and WCA (whole water, chilled, 

acidified) bottle types—provided that they have been 

processed according to USGS field manual protocols 

(Wilde and others, 1998). Second, nitrogen and 

phosphorus are recovered quantitatively from digests 

prepared by the method described in this report as 

explained in section 2.2. Furthermore, manual postdigestion pH adjustment prior to colorimetric 

determinations required by other previously published 

alkaline persulfate digestion methods (Valderrama, 

1981; Hosomi and Sudo, 1986; D’Elia and others, 

1987; Ameel and others, 1993; D’Elia and others, 

1997) is not necessary in the method described in this 

report. This modification reduces digest preparation 
Introduction 3

time substantially. Hopefully, methodological 

improvements and comparative data in this report in 

concert with publication of Standard Methods method 

4500-N C will encourage analysts and regulators to 

consider potential benefits of more widespread 

application of alkaline persulfate digestion as an 

alternative to Kjeldahl digestion for nitrogen and 

phosphorus determinations in freshwater regimes.

This report provides complete details of the largescale and geographically diverse study conducted by 

the USGS between October 1, 2001, and September 30, 

2002, to evaluate and validate alkaline persulfate 

digestion as a more sensitive, accurate, and less toxic 

alternative to Kjeldahl digestion for routine 

determination of nitrogen and phosphorus in surfaceand ground-water samples.

Purpose and Scope

This report describes USGS methods I-2650-03 

and I-4650-03 for determining total nitrogen and total 

phosphorus in filtered and whole-water alkaline 

persulfate digests, respectively. All aspects of the 

methods are described, including sample preparation 

and digestion, colorimetric determinations of nitrate 

and orthophosphate in alkaline persulfate digests, 

calculation of results, bias, precision, and repeatability 

of results, and conventions for reporting results. These 

methods supplement other methods of the USGS for 

determination of inorganic substances in water that are 

described by Fishman and Friedman (1989) and 

Fishman (1993). Primary objectives of this study were 

as follows:

1. To eliminate hazards and toxic wastes associated 

with Kjeldahl nitrogen and Kjeldahl 

phosphorus determinations.

2. To ascertain if and under what conditions 

alkaline persulfate digestion methods can be 

applied to samples preserved by acidification.

3. To develop an alkaline persulfate digestion 

procedure that is amenable to automation and 

less labor intensive than existing Kjeldahl 

digestion procedures.

4. To achieve lower detection limits for total and 

dissolved nitrogen than can be achieved by 

typical Kjeldahl digestion methods.

5. To evaluate statistical equivalence of dissolved 

and total nitrogen concentrations determined 

by Kjeldahl and alkaline persulfate 

digestion methods

6. To evaluate statistical equivalence of dissolved 

and total phosphorus concentrations 

determined by Kjeldahl and alkaline 

persulfate digestion methods.

7. To establish guidelines for interpreting 

dissolved and total nitrogen and phosphorus 

concentrations that result from alkaline 

persulfate digestion in relation to those that 

result from Kjeldahl digestion.

8. To verify that alkaline persulfate digestion is a 

more sensitive, accurate, and environmentally responsible alternative to Kjeldahl 

digestion for routine, simultaneous 

determination of nitrogen and phosphorus in 

surface and ground water—the conclusion of 

several previously published, smaller scale 

studies—on the basis of a large, 

geographically and seasonally diverse data 

set and to demonstrate the method's 

applicability for compliance monitoring and 

water-quality assessment studies.

Acknowledgments

The authors gratefully acknowledge Carolyn Keefe 

and Carl Zimmermann at the Chesapeake Biological 

Laboratory Nutrient Analytical Services Laboratory 

(NASL) in Solomons, Md., who informally reviewed a 

preliminary version of this report. They also kindly 

shared technical and operational details of NASL's 

long-established alkaline persulfate digestion nitrogen 

and phosphorus methods that have been widely applied 

in nutrient studies of the Chesapeake Bay. We also 

thank Richard Axler and John Ameel at the University 

of Minnesota–Duluth Center for Water and the 

Environment—major participants in validation of the 

alkaline persulfate digestion method for determination 

of dissolved and total nitrogen published in the 20th 

edition of Standard Methods—who reviewed this 

report. Their comments were consistently insightful 

and helped us focus the Introduction and Conclusions 

sections. Tom Maddox at the University of Georgia 

Stable Isotope Laboratory in Athens also provided 

helpful discussions and several literature citations 

about applying alkaline persulfate digestion methods to 

soil and sediment analysis.
4 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

Ron Antweiler, USGS National Research Program, 

provided helpful discussions about applying t-tests to 

statistical method comparison data in this report. Tom 

Bushly and Richard Husband, Information Technology 

Section at the USGS National Water Quality 

Laboratory (NWQL) performed several large data-base 

queries and designed custom reports that streamlined 

sample selection and data analysis. This report also 

benefited from technical reviews by Colleen Gupta and 

Mary Cast (USGS, NWQL) and Peter Rogerson 

(USGS, Office of Water Quality). We also thank 

Jon W. Raese (USGS, NWQL) for editorial review and 

Barbara L. Kemp (USGS, NWQL) for manuscript 

preparation.

ANALYTICAL METHOD

Inorganic Constituents and Parameter Codes 

(see table 1): Nitrogen and phosphorus, total 

dissolved, I-2650-03 (mg/L as N or P); nitrogen 

and phosphorus, total whole water, I-4650-03 

(mg/L as N or P)

1. Application

These methods are intended for determination of 

total nitrogen (organic nitrogen + ammonium + nitrate 

+ nitrite) and phosphorus (all forms) in filtered and 

whole-water samples by alkaline persulfate digestion. 

They were validated for determination of total nitrogen 

and total phosphorus in drinking water, wastewater, 

and water-suspended sediment. Their applicability to 

bottom materials was not investigated. Analytical 

ranges are 0.03 to 5.00 mg-N/L for dissolved and total 

nitrogen and 0.01 to 2.00 mg-P/L for dissolved and 

total phosphorus.

2. Method Summary and Analytical 

Considerations

2.1 Filtered and whole-water samples are 

dispensed into glass culture tubes, dosed with alkaline 

persulfate reagent, capped tightly, and digested in an 

autoclave at 250oF (121oC) and 17 lb/in2 (117.2 kPa) for 

1 hour. The alkaline persulfate digestion procedure 

oxidizes all forms of inorganic and organic nitrogen to 

nitrate and hydrolyzes all forms of inorganic and 

organic phosphorus to orthophosphate. Nitrate and 

orthophosphate in alkaline persulfate digests are 

determined in parallel with a 2-channel photometric, 

air-segmented continuous flow analyzer.

2.2 Digest preparation protocols and reagent 

formulations were adapted from previously published 

procedures (Valderrama, 1981; Hosomi and Sudo, 

1986; Ameel and others, 1993; D’Elia and others, 

1997; American Public Health Association, 1998b). 

Two other reports (Nydahl, 1978; Cabrera and Beare, 

1993) provided insight into the potential for low 

nitrogen recovery in samples containing high 

concentrations of dissolved and particulate organic 

carbon.

Quantitative recovery of nitrogen and phosphorus 

by alkaline persulfate digestion depends critically on a 

Table 1. Laboratory, parameter, and method codes for U.S. Geological Survey alkaline persulfate digestion 

total nitrogen and total phosphorus methods I-2650-03 and I-4650-03

[Lab, laboratory; FCC, filtered chilled container; FCA, filtered, chilled, acidified; WCA, whole water, chilled, acidified; 

μm, micrometer; mL, milliliter; USGS, U.S. Geological Survey] 

 Codes 

Description Lab Parameter Method 

Bottle 

type 

Nitrogen, total dissolved, alkaline persulfate digestion......................... 2754 62854 A FCC1

Nitrogen, total dissolved, alkaline persulfate digestion, acidified........ 2755 62854 B FCA2

Nitrogen, total whole-water, alkaline persulfate digestion, acidified.... 2756 62855 A WCA2

Phosphorus, total dissolved, alkaline persulfate digestion.................... 2757 00666 I FCC1

Phosphorus, total dissolved, alkaline persulfate digestion, acidified.... 2758 00666 J FCA2

Phosphorus, total whole-water, alkaline persulfate digestion, acidified 2759 00665 H WCA2

 1FCC samples must be processed through 0.45-μm filters at collection sites.

 2

FCA and WCA samples must be amended with 1 mL of 4.5 N H2SO4 solution (USGS water-quality field supply number 

Q438FLD) per 120 mL of sample at collection sites.
Analytical Method 5

progressive decrease in pH (initial pH >12, final 

pH ≤ 2.2) during the 1-hour course of the digestion 

(Hosomi and Sudo, 1986). These dynamic reaction 

conditions are achieved by formulating the digestion 

reagent with approximately equimolar concentrations 

of persulfate and hydroxide ions—0.05 M, initial pH 

>12 after 1 + 2 dilution by samples in this method. 

Under these initially alkaline conditions, dissolved and 

suspended nitrogen in samples oxidize to nitrate. As 

the digestion proceeds, bisulfate ions resulting from 

thermal decomposition of persulfate first neutralize and 

then acidify the reaction mixture by the following 

chemical reaction:

After all of the persulfate has decomposed, the 

digest mixture pH approaches 2, and under these acidic 

conditions, dissolved and suspended phosphorus 

hydrolyze to orthophosphate. The foregoing 

discussion indicates that analysis of samples with 

variable and unknown acidity or alkalinity by alkaline 

persulfate digestion methods will be problematic. 

Users of this method are cautioned that amending FCA 

and WCA samples with concentrations of sulfuric acid 

other than those specified in USGS field manual 

protocols (Wilde and others, 1998) likely will result in 

undetected method failure and possible reporting of 

erroneous results. See section 3.1.4 of this report for 

additional details.

As is the case for Kjeldahl digestion, alkaline 

persulfate digestion converts all forms of phosphorus 

to orthophosphate. Thus alkaline persulfate digestion 

dissolved and total phosphorus (DPAlkP and TPAlkP) 

concentrations can be compared directly with Kjeldahl 

digestion dissolved and total phosphorus (KDP and 

KTP) concentrations by graphical and statistical 

analysis. This is not the case, however, for Kjeldahl 

dissolved and total nitrogen (KDN and KTN) 

concentrations and alkaline persulfate digestion 

dissolved and total nitrogen (DNAlkP and TNAlkP) 

concentrations. In principle, organic nitrogen, but not 

nitrate or nitrite, is reduced to ammonium during 

Kjeldahl digestion. Determining ammonium in 

Kjeldahl digests, therefore, measures organic nitrogen 

+ ammonium. Alkaline persulfate digestion oxidizes 

all forms of nitrogen to nitrate. Determining nitrate + 

nitrite in alkaline persulfate digests, therefore, 

measures total nitrogen (organic nitrogen + ammonium 

+ nitrite + nitrate). To reconcile this difference 

between the two methods, nitrate + nitrite 

concentrations were subtracted from DNAlkP and 

TNAlkP concentrations prior to graphical and statistical 

comparisons with KDN and KTN concentrations 

throughout this report. For this purpose and as a 

quality-control (QC) check, all filtered and wholewater samples selected for alkaline persulfate digestion 

also were analyzed for dissolved nitrate + nitrite, 

ammonium, and orthophosphate on the same day that 

digests were prepared. Particulates were removed from 

acidified, whole-water samples (WCA bottle type) by 

0.45-μm filtration prior to dissolved nutrient 

determinations, as described in section 4.6 of this 

report.

2.3 A 2-channel, air-segmented continuous flow 

analyzer was configured for simultaneous photometric 

determination of nitrate + nitrite and orthophosphate in 

alkaline persulfate digests. Nitrate + nitrite was 

determined by a cadmium-reduction, Griess-reaction 

method (Wood and others, 1967) equivalent to U.S. 

Environmental Protection Agency (USEPA) method 

353.2 (U.S. Environmental Protection Agency, 1993) 

and U.S. Geological Survey (USGS) method I-2545-90 

(Fishman, 1993, p. 157) except that sulfanilamide and 

N-(1-naphthy)ethylenediamine reagents were separate 

rather than combined. The analytical cartridge diagram 

is shown in figure 1. Orthophosphate was determined 

by a phosphoantimonylmolybdenum blue method 

(Murphy and Riley, 1962; Pai and others, 1990), which 

is equivalent to the 2-reagent variants (separate 

molybdate and ascorbic acid reagents) of USEPA 

method 365.1 (U.S. Environmental Protection Agency, 

1993) and USGS method I-2601-90 (Fishman, 1993). 

The analytical cartridge diagram is shown in figure 2.

3. Interferences

3.1 Alkaline Persulfate Digestion

3.1.1 Chloride concentrations up to 1,000 

mg/L (the highest tested for this report) do not 

interfere. Furthermore, because good results are 

obtained for seawater in 2 + 1 mixture with digestion 

reagent (D’Elia and others, 1997), chloride 

concentrations of about 10,000 mg/L apparently are 

tolerated provided that calibrants are matrix matched. 

Higher chloride concentrations, however, are likely to 

interfere because of reaction with persulfate to form 

oxychlorides or chlorine that might deplete persulfate 

required to oxidize inorganic and organic nitrogen 

2- - 1

28 2 4 2 2 S O + H O 2 HSO + O ⎯⎯→
 6 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER FLOW RATE color code (milliliters per minute) *A 5 mg-N/L nitrate plus nitrite calibrant produces a peak height of about 90 percent of full scale when the 

photometer standard calibration control is set at ≈ 1.1.

5-turn

coil

10-turn

coil

25-turn

coil

Air

SAN

NED

Wash

Solution

Air

Buffer

Sample

10-millimeter*

flow cell

540 nanometers

Waste 1 PBCdR

Debubbler

3 1

orange/yellow

(0.118)

orange/orange

(0.287)

orange/yellow

(0.118)

orange/green

(0.074)

yellow/blue

(0.722)

orange/green

(0.074)

orange/green

(0.074)

PUMP

green/green

(0.947)

2

123

EXPLANATION

Air bar, 90 bubbles per minuteDual injection fitting, P/N 303-0107Debubbler fitting, P/N 303-0103Reagent addition tee, P/N 303-0102

Figure 1. Analytical cartridge diagram for the air-segmented continuous flow analyzer (Alpkem RFA-300) used to automate photometric determination of nitrate + nitrite in alkaline persulfate digests with a cadmium-reduction, Griess reaction method.
Analytical Method 7

FLOW RATE

color code

(milliliters per minute)

5-turn

coil

5-turn

coil

5-turn

coil

37 °C

2 milliliters

Air

Ascorbic Acid

Wash Solution

SLS Diluent

Sample

Acid Molybdate Insulated 15-millimeter*

flow cell

880 nanometers

Waste 1 2 2 3

orange/yellow

(0.118)

orange/green

(0.074)

orange/green

(0.074)

green/green

(0.947)

black/black

(0.226)

orange/white

(0.166)

*A 2 mg-P/L orthophosphate calibrant produces a peak height 

of about 90 percent of full scale when the photometer standard calibration control is set at ≈ 1.5. 

12

EXPLANATION

Air bar, 90 bubbles per minuteDual injection fitting, P/N 303-0107Reagent addition tee, P/N 303-0102

3 The 0.034-inch (0.086 cm) inside diameter (i.d.) poly-

 ethylene tubing that connects the heat bath coil outlet to the flow cell is thermally insulated by sheathing it with a piece of 0.10-inch (0.25 cm) i.d. polyvinyl chloride transmission tubing. Also the flow cell barrel is thermally insulated by stretch- wrapping it with several layers of thin foam plastic.

Figure 2. Analytical cartridge diagram for the air-segmented continuous flow analyzer (Alpkem RFA-300) used to automate photometric determination of orthophosphate in alkaline persulfate digests with a reduced phosphoantimonylmolybdenum blue reaction method.
8 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

species to nitrate. Resulting active chlorine species 

also can interfere in colorimetric reactions used to 

determine nitrate and orthophosphate in digests.

3.1.2 Sulfate concentrations up to 1,000 

mg/L (the highest tested for this report) do not 

interfere.

3.1.3 Organic carbon concentrations greater 

than 150 mg/L interfere because of reaction with 

persulfate to form carbon dioxide, thus depleting 

persulfate required to oxidize inorganic and organic 

nitrogen species to nitrate.

3.1.4 Overacidification of FCA and WCA 

samples at collection sites can result in low recovery of 

inorganic and organic nitrogen at the NWQL. The 

possibility of overacidification can be avoided by 

exclusive use of the sulfuric acid field-amendment 

solution—one vial containing 1 mL of 4.5 N H2SO4

(One Stop Shopping number FLD-438) per 120 mL of 

sample—which is specified in the USGS National 

Field Manual (Wilde and others, 1998). See the first 

note in section 6.1 of this report for additional details.

3.1.5 Nitrate and nitrite do not contribute to 

KDN and KTN concentrations in principle, but in 

practice, positive and negative interferences by these 

ions are well known—see, for example, American 

Public Health Association, 1998c; Patton and Truitt, 

2000. This interference can confound comparison of 

KN and NAlkP concentrations when dissolved nitrate 

concentrations are greater than about 0.1 mg 

NO3

-

-N/L.

3.1.6 Suspended particles remaining in 

digests must be removed by sedimentation and 

decantation or filtration prior to colorimetric analyses.

3.2 Colorimetric Nitrate + Nitrite Determination

3.2.1 Typically, concentrations of 

substances with potential to interfere in cadmiumreduction, Griess-reaction nitrate + nitrite methods are 

negligible in ambient surface- and ground-water 

samples. For specific details of inorganic and organic 

compounds that might interfere in the color reaction, 

see Norwitz and Keliher (1985, 1986), as well as more 

general information by the American Public Health 

Association (1998a).

3.2.2 Sulfides, which are often present in 

anoxic water and well known to deactivate cadmium 

reduction reactors, are oxidized during the alkaline 

persulfate digestion and are unlikely to interfere.

3.3 Colorimetric Orthophosphate Determination

3.3.1 Barium, lead, and silver can interfere 

by forming insoluble phosphates, but their 

concentrations in natural-water samples usually are 

less than the interference threshold (Fishman, 1993)

3.3.2 Interference from silicate, which also 

can form reduced heteropoly acids with molybdenum 

(Zhang and others, 1999), is negligible under reaction 

conditions used for this report.

3.3.3 Arsenate, AsO4

3-—but not arsenite, 

AsO3

3-—can interfere by forming reduced heteropoly 

acids analogous to those formed by orthophosphate 

(Johnson, 1971). Because of the possibility that 

arsenite might be oxidized to arsenate by persulfate, 

both species at concentrations up to 20 mg-As/L in 

deionized water were digested and analyzed. With 

reference to table 2, it is apparent that a major fraction 

of arsenite is oxidized to arsenate during alkaline 

persulfate digestion and that interference by either 

species up to 1 mg-As/L is negligible.

4. Instrumentation and Auxiliary 

Analyses

4.1 RFA-300TM, third-generation, air-segmented 

continuous flow analyzers (Alpkem) were used to 

automate photometric determination of nitrate + nitrite 

and orthophosphate in alkaline persulfate digests and 

dissolved ammonium, nitrate + nitrite, and 

orthophosphate in filtered- and whole-water samples 

prior to digestion. Modules in these systems include 

301 samplers, 302 peristaltic pumps, 313 analytical 

cartridge bases, 314 power modules, 305A 

photometers, and a personal computer (PC)-based data 

acquisition and processing system. Alternative 

instrumentation—flow injection analyzers, sequential 

injection analyzers, other second- or third-generation 

continuous flow analyzers, or automated batch 

analyzers—also could be used to automate photometric 

finishes.

Table 2. Data from a study of arsenate and arsenite interference 

in alkaline persulfate total phosphorus determinations 

[mg-As/L, milligrams of arsenic per liter; mg-P/L, milligrams of 

phosphorus per liter; nd, not detected; ≈, nearly equal to; ±, plus or minus] 

AsO4

3-

added 

mg-As/L 

PO4

3-

found 

mg-P/L 

AsO3

3-

added 

mg-As/L 

PO4

3-

found 

mg-P/L 

0.5 nd 0.5 nd 

1.0 nd 1.0 nd 

2.0 ≈ 0.05 2.0 nd 

5.0 0.32 ± 0.01 5.0 0.29 ± 0.04 

10.0 1.14 ± 0.13 10.0 0.91 ± 0.06 

20.0 off scale 20.0 off scale 
Analytical Method 9

4.2 Photometric data were acquired and 

processed automatically using FASPacTM version 1.34 

software (Astoria-Pacific, Clackamas, Ore.). This 

software operates under Microsoft Windows on a PC 

platform and includes a model 350 interface box that 

controls the sampler and digitizes analog photometer 

outputs with 16-bit resolution. Other data acquisition 

systems could be used provided that the A/D converter 

has 16-bit resolution and is capable of acquiring data at 

frequencies ranging from 0.5 to 2 Hz, that is, from 30 

points/min to 120 points/min. As a general rule, data 

acquisition frequencies for air-segmented continuous 

flow analyzers should match the roller lift-off 

frequency of the peristaltic pump (Patton and Wade, 

1997), that is, 0.5 Hz for Technicon AutoAnalyzer II TM 

and 1.5 Hz for Alpkem RFA-300 equipment. Data 

acquisition frequencies in the range of 2 to 5 Hz are 

suitable for photometric flow-injection analyzers.

4.3 Operating characteristics for this equipment 

are listed in table 3.

4.4 Dissolved ammonium, nitrate + nitrite, and 

orthophosphate in undigested samples were 

determined photometrically by USGS automated 

continuous flow methods I-2522-90, I-2545-90 

(2-reagent variant), and I-2601-90 (2-reagent variant), 

respectively. These methods are described in Fishman 

(1993).

4.5 The pH of WCA samples was estimated with 

narrow range (0–2.5) colorimetric pH-indicating test 

strips to detect improperly acidified samples that had 

pH values outside the expected range of 1.6 to 1.9.

4.6 WCA samples were processed through 5-mL 

capacity UniPrepTM syringeless filters equipped with 

0.45-μm nylon membranes (Whatman, Clifton, N.J.) to 

remove suspended solids prior to determination of 

dissolved ammonium, nitrate + nitrite, and 

orthophosphate. These syringeless filters also were 

used to remove suspended solids from WCA-sample 

digests prior to photometric analysis when simple 

sedimentation and decantation into analyzer cups failed 

to do so.

5. Apparatus

5.1 Samples were digested in an autoclave 

(model number STME, Market Forge Industries, Inc., 

Everett, Mass.) operated at 250oF (121oC) and 17 lb/in2 

(117.2 kPa) for 1 hour.

5.2 Filtered and chilled sample (FCC bottle type) 

digests were prepared robotically using a large-scale, 

syringe-pump-based x-y-z sample dispenser/diluter 

module (model number ML-4200, Hamilton Company, 

Reno, Nev.). This system is equipped with four probes 

and four 10-mL syringe pumps that operate in tandem 

under control of DOS-based EclipseTM software 

(Hamilton Company, Reno, Nev.). Custom 

modifications to the ML-4200 system, including a 

pneumatically actuated probe expander, fixtures, and a 

variety of bottle and test-tube racks, were obtained 

from another vendor (Robotics Plus, Houston, Tex.).

5.3 Whole-water (WCA bottle type) sample 

digests were prepared manually using EDP PlusTM 

Table 3. Settings and operational details of Alpkem RFA-300 continuous flow analyzers used for this study

[nm, nanometer; mm, millimeter; mg-N/L, milligrams nitrogen per liter; mg-P/L, milligrams phosphorus per liter; 

≈, nearly equal to; min, minute; mL, milliliter; –, not applicable; °C, degrees Celsius; s, second; h, hour] 

Instrumental conditions Nitrate + nitrite Orthophosphate 

Analytical wavelength 540 nm 880 nm 

Flow cell path length 10 mm 15 mm 

Calibration range 0.05 to 5.0 mg-N/L 0.01 to 2.0 mg-P/L 

Standard calibration control setting ≈1.1 ≈1.5 

Segmentation rate (bubbles min-1) 90 90 

Heated reaction coil volume None used 2 mL 

Heated reaction coil temperature – 37°C

Dwell time (seconds) 140 260 

Sample time (volume) 25 s (95 μL) 25 s (31 μL) 

Wash time (volume) 10 s (38 μL) 10 s (12 μL) 

Analysis rate, sample-to-wash ratio ≈103/h, 5:2 ≈103/h, 5:2 
10 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

electronic, digital pipets (Rainin Instruments, 

Emeryville, Calif.) equipped with a 10-mL liquid end.

5.4 Digestion vessels were 20 x 150 mm Pyrex®, 

screw-cap culture tubes (VWR 53283-810; Fisher 

14-957-76E or 14-959-37C; or equivalent), and 

18-415 linerless polypropylene caps (Comair Glass, 

Inc., Vineland, N.J.—Part number 14-0441-004).

6. Reagents

This section provides detailed instructions for 

preparing digestion and colorimetric reagents. All 

references to deionized water (DI) refer to NWQL inhouse DI water, which is equivalent to ASTM type I DI 

water (American Society for Testing and Materials, 

2001, p. 107–109) for nutrient analysis. All volumetric 

glassware and reagent and calibrant storage containers 

should be triple rinsed with dilute (≈5 percent v/v) 

hydrochloric acid and DI water just prior to use. 

Additionally storage containers for reagents and 

calibrants should be triple rinsed with small portions of 

the solutions before they are filled.

6.1 Digestion Reagents

NOTE: The alkaline persulfate digestion reagent for 

FCA and WCA samples (section 6.1.4) contains an 

additional amount of sodium hydroxide that is 

calculated to neutralize the sulfuric acid added to these 

samples at collection sites.

6.1.1 Sodium hydroxide, 1.5 M (for FCC 

samples): Dissolve 60 g of sodium hydroxide (NaOH, 

FW=40.0) in about 800 mL of DI water in a 1-L 

volumetric flask. [Caution: When NaOH dissolves in 

water, heat is released.] After dissolution is complete, 

allow the resulting solution to cool and dilute it to the 

mark with DI water. Transfer this reagent to a plastic 

bottle in which it is stable at room temperature for 6 

months.

6.1.2 Sodium hydroxide, 2.3 M (for FCA 

and WCA samples): Dissolve 92 g of sodium 

hydroxide (NaOH, FW=40.0) in about 800 mL of DI 

water in a 1-L volumetric flask. [See caution in 6.1.1.] 

After dissolution is complete, allow the resulting 

solution to cool and dilute it to the mark with DI water. 

Transfer this reagent to a plastic bottle in which it is 

stable at room temperature for 6 months.

6.1.3 Alkaline persulfate digestion reagent 

(for FCC samples): Add 18.0 g of potassium persulfate 

(K2S2O8, FW=270.33) and 45 mL of 1.5 M sodium 

hydroxide solution to about 350 mL of DI water in a 

graduated 500-mL PyrexTM media bottle (Corning 

number 1395-500 or equivalent). Cap the bottle, swirl 

its contents, and place it in an ultrasonic bath until 

potassium persulfate dissolution is complete (about 10 

minutes). Remove the bottle from the ultrasonic bath, 

dry its outer surfaces, and then add enough DI water to 

bring the volume to 450 mL. (Make a line on the side 

of the bottle that indicates this volume to within 

±5 mL.) Swirl the bottle to mix its contents and then 

divide the resulting solution among four, 125-mL clear 

plastic bottles used with the robotic digest preparation 

system. Prepare this reagent daily.

6.1.4 Alkaline persulfate digestion reagent 

(for FCA and WCA samples): Add 18.0 g of potassium 

persulfate (K2S2O8, FW=270.33) and 45 mL of 2.3 M 

sodium hydroxide solution to about 350 mL of DI 

water in a graduated 500-mL PyrexTM media bottle 

(Corning number 1395-500 or equivalent). Then 

complete preparation of this reagent exactly as 

described in 6.1.3. Prepare this reagent daily.

NOTE: Reagent volumes in 6.1.3 and 6.1.4 (450 mL) 

are sufficient to prepare 80 digests plus a 15-percent 

excess for rinsing and providing a liquid level in the 

125-mL bottles necessary to prevent air aspiration 

during robotic dispensing operations. For manual 

digest preparation, a 400-mL volume of digestion 

reagent should be sufficient.

6.2 Colorimetric Reagents

6.2.1 Sampler wash reservoir solution (0.05 

M sodium bisulfate): Dissolve 6.9 g of sodium 

bisulfate (NaHSO4•H2O, FW=138.08) in about 800 mL 

of DI water in a graduated 1-L PyrexTM media bottle. 

Dilute this solution to the mark with DI water, mix it 

well, and store it tightly capped at room temperature.

NOTE: This solution matches the matrix of sample 

digests. Use it as the matrix for continuing calibration 

verification (CCV) solutions and any other undigested 

check samples.

6.3 Orthophosphate Determination

6.3.1 Stock potassium antimony tartrate 

reagent: Dissolve 3.0 g of antimony potassium tartrate 

[K(SbO)C4H4O7•1⁄2 H2O, FW=333.93] in about 800 

mL of DI water in a 1-L volumetric flask. Dilute this 

solution to the mark with DI water and mix it well. 
Analytical Method 11

Transfer this reagent to a plastic bottle in which it is 

stable for 6 months at room temperature.

6.3.2 Stock ascorbic acid reagent: Dissolve 

4.5 g of ascorbic acid (C6H8O6, FW=176.1) in about 

200 mL of DI water in a 250-mL volumetric flask. 

Dilute this solution to the mark with DI water, mix it 

well, and transfer to a 250-mL glass bottle that has been 

previously rinsed with 5 percent (v/v) hydrochloric 

acid solution and DI water. This reagent is stable for 2 

weeks at 4°C.

6.3.3 Stock sodium lauryl sulfate reagent 

(15 percent w/w): Add 340 mL of DI water to 60 g of 

sodium lauryl sulfate [SLS, CH3(CH2)11OSO3Na, 

FW=288.38] in a 500-mL PyrexTM media bottle. Cap 

the bottle and place it in an ultrasonic bath until the SLS 

dissolves completely (about 30 minutes). Manual 

inversion of the bottle at 5-minute intervals speeds 

dissolution. Transfer this solution to a plastic bottle in 

which it is stable indefinitely at room temperature.

6.3.4 Acidic molybdate-antimony reagent: 

Using a graduated cylinder, cautiously add 72 mL of 

concentrated sulfuric acid (H2SO4, sp. gr. 1.84) to about 

700 mL of DI water in a 1-L volumetric flask. Work in 

a hood and manually swirl or magnetically stir the flask 

during each addition of sulfuric acid. Next add 7.7 g of 

ammonium molybdate [(NH4)6Mo7O24•4H2O, 

FW=1235.86] to the hot sulfuric acid solution. 

Manually swirl or magnetically stir the contents of the 

flask until the ammonium molybdate dissolves. Then 

add 50 mL of stock antimony potassium tartrate 

solution (6.3.1) and again mix the contents of the flask 

thoroughly. After the resulting solution has cooled, 

dilute it to the mark with DI water, mix it well, and 

transfer it to a clean 1-L plastic bottle in which it is 

stable for 1 year at room temperature.

6.3.5 Sodium lauryl sulfate diluent reagent: 

Use a 100-mL graduated cylinder to dispense 10 mL of 

stock SLS (6.3.3) and 90 mL of DI water into a small 

plastic bottle. Manually swirl the bottle to mix its 

contents. Prepare this reagent daily.

6.3.6 Ascorbic acid reagent: Use a 50-mL 

graduated cylinder to dispense 5 mL of the stock 

ascorbic acid reagent (6.3.2) and 25 mL of DI water 

into an amber glass reagent bottle. Manually swirl the 

bottle to mix its contents. Prepare this solution daily.

6.3.7 Startup/shutdown solution: Add 1 mL 

of stock SLS reagent to 100 mL of DI water in a small 

plastic bottle. Thoroughly rinse the bottle and prepare 

a fresh solution every few days or as needed.

6.4 Nitrate Determination

6.4.1 Copper (II) sulfate reagent (2 percent w/v): 

Dissolve 20 g of copper sulfate pentahydrate 

(CuSO4•5H2O, FW=249.7) in about 800 mL of DI 

water in a 1-L volumetric flask. Dilute this solution to 

the mark with DI water, mix it well, and transfer it to a 

1-L plastic bottle. This reagent is stable for several 

years at room temperature.

6.4.2 Imidazole buffer, 0.1 M, (pH 7.5): In a 

hood, cautiously add 5.0 mL of concentrated 

hydrochloric acid (HCl, ∼12 M) and 1.0 mL of 2 

percent copper sulfate solution to 1,600 mL of DI water 

in a 2-L volumetric flask. Mix the contents of the flask 

thoroughly and then add 13.6 g of imidazole (C3H4N2, 

FW=68.08). Again swirl or shake the flask until the 

imidazole dissolves. Dilute the resulting solution to the 

mark with DI water, mix it well, and transfer it into two 

1-L plastic bottles. This reagent is stable for 6 months 

at room temperature. 

NOTE: Add 250 μL of Brij-35 surfactant to 250 mL 

of imidazole buffer each time its container is refilled on 

the continuous flow analyzer. Do not add Brij-35 to the 

bulk buffer solution.

6.4.3 Packed bed cadmium reactor: 

Cadmium reactors are prepared by slurry packing 40- 

to 60-mesh, copperized cadmium granules into 6-cm 

lengths of PTFE TeflonTM tubing (1.6 mm i.d. × 3.2 mm 

o.d.). Cadmium granules are retained in the column 

with hydrophilic plastic frits (40-μm nominal pore 

size). Detailed instructions for preparing copperized 

cadmium granules and packing them into columns can 

be found in NWQL standard operating procedure 

(SOP) IM0384.0 (or subsequent revisions; available on 

request).

6.4.4 Sulfanilamide reagent (“SAN”): Use a 

graduated cylinder to dispense 100 mL of concentrated 

hydrochloric acid (HCl, 36.5–38.0 percent, ≈12 M) 

into about 700 mL of DI water in a 1-L volumetric 

flask. Work in a hood and manually swirl or 

magnetically stir the flask during each addition of HCl. 

Add 10.0 g of SAN (C6H8N2O2S, FW=172.20) to the 

warm hydrochloric acid solution. Manually shake, 

sonicate, or magnetically stir the contents of the flask 

until the SAN dissolves. After the resulting solution 

has cooled, dilute it to the mark with DI water, mix it 

well, and transfer it to a clean 1-L plastic bottle in 

which it is stable for 1 year at room temperature.

6.4.5 N-(1-Naphthyl)ethylenediamine 

dihydrochloride reagent (“NED”): Dissolve 1.0 g 
12 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

NED (C12H14N2•2HCl, FW=259.2) in about 800 mL of 

DI water in a 1-L volumetric flask. Dilute the resulting 

solution to the mark with DI water and mix well by 

manually shaking the flask. Transfer this reagent to a 

1-L amber glass bottle in which it is stable for 6 months 

at room temperature.

6.4.6 Startup/shutdown solution: Add 

250 μL of Brij-35 surfactant to 250 mL of DI water in 

a plastic bottle. Thoroughly rinse the bottle and 

prepare a fresh solution every few days or as needed.

7. Calibrants and Quality-Control 

Solutions

This section provides detailed instructions for 

preparing calibrants, matrix spike solution, qualitycontrol check solutions, and digestion check solution.

7.1 Potassium nitrate stock calibrant solution, 

1 mL =2.5 mg-N: Dissolve 1.805 g of potassium nitrate 

(KNO3, FW=101.1) in about 80 mL of DI water in a 

100-mL volumetric flask. Dilute this solution to the 

mark with DI water and mix it thoroughly by manual 

inversion and shaking. Transfer the stock calibrant to a 

100-mL PyrexTM media bottle in which it is stable for 6 

months at 4°C.

7.2 Potassium di-hydrogen phosphate stock 

calibrant solution, 1 mL =1.0 mg-P: Dissolve 0.4394 g 

potassium di-hydrogen phosphate (KH2PO4, 

FW=136.09) in about 80 mL of DI water in a 100-mL 

volumetric flask. Dilute this solution to the mark with 

DI water and mix it thoroughly by manual inversion 

and shaking. Transfer the stock calibrant to a 100-mL 

PyrexTM media bottle in which it is stable for 6 months 

at 4°C.

7.3 Sulfuric acid ≈1.8 M: Use a 25-mL 

graduated cylinder to dispense 10 mL of concentrated 

sulfuric acid (H2SO4, sp. gr. 1.84) into about 75 mL of 

DI water in a 100-mL volumetric flask. After the 

solution cools, dilute it to the mark with DI water, mix 

it well, and transfer it to a 125-mL plastic bottle. Make 

a new batch of this acid each time acidified working 

calibrants and blanks are prepared and use the 

remainder to prepare acidified blank solution as 

needed.

7.4 Mixed stock calibrant solution, 1 mL = 1.25 

mg-N and 0.5 mg-P: Dispense equal volumes 

(minimum of 2 mL each) of nitrate (7.1) and phosphate 

(7.2) stock calibrants into a small beaker and mix them 

thoroughly. Prepare this solution each time working 

calibrants are prepared.

7.5 Working calibrant solutions (for FCC 

samples): Use two adjustable, digital pipets (ranges 10 

to 100 μL and 100 to 1,000 μL) to dispense the volumes 

of mixed stock calibrant (7.4) listed in table 4 into 

250-mL volumetric flasks that each contain about 200 

mL of DI water. Dilute the working calibrants to the 

mark with DI water and mix them thoroughly by 

manual inversion and shaking. Transfer the working 

calibrants to 250-mL PyrexTM media bottles in which 

they are stable for 4 weeks at 4°C.

7.6 Acidified working calibrant solutions (for 

FCA and WCA samples): Prepare these calibrants 

Table 4. Volumes of mixed calibrant and amendment solution required to prepare 

working calibrants and blanks for determination of total nitrogen and phosphorus by the 

alkaline persulfate digestion method. Final volumes are 250 mL 

[μL, microliter; mL, milliliter; mg-N/L, milligrams nitrogen per liter; mg-P/L, milligrams 

phosphorus per liter; M, molarity (moles per liter); FCA, filtered, chilled, acidified (bottle type); 

WCA, whole water, chilled, acidified (bottle type)] 

Calibrant 

identity 

Mixed 

calibrant 

volume (μL) 

Volume 

1.8 M H2SO4 1 (mL) 

Nominal 

concentration 

(mg-N/L) 

Nominal 

concentration 

(mg-P/L) 

C1 1,000 2.5 5.00 2.00 

C2 750 2.5 3.75 1.50 

C3 500 2.5 2.50 1.00 

C4 250 2.5 1.25 0.50 

C5 100 2.5 0.50 0.20 

C6 2

 6 2 2.5 0.03 0.012 

C7 0 2.5 0 0 1Add H2

SO4 only to acidified calibrants as described in section 7.6. 2Prepare 1 L of C6 (24 μL of mixed calibrant and 10 mL of 1.8 M H2

SO4, if appropriate, diluted 

to 1 L with DI water) to minimize dispensing error.
Analytical Method 13

identically to those described in section 7.5, except add 

2.5 mL of 1.8 M H2SO4 to each flask before diluting it 

to the mark with DI water.

7.7 Check standards (for FCC samples): Check 

standards in three concentration ranges, which were 

designated Low, High, and Very high, were prepared 

from a concentrated commercial nutrient QC mixture 

(DemandTM, Environmental Resource Associates, 

Arvada, Colo.), as listed in table 5. Transfer check 

standards to 1-L PyrexTM media bottles in which they 

are stable for 2 months at 4oC. Each of these check 

standards was dispensed, digested, and analyzed along 

with every batch of filtered and whole-water samples 

analyzed for this study.

7.8 Acidified check standards (for FCA and WCA 

samples): Prepare these check standards identically to 

those described in section 7.7, except add 10.0 mL of 

1.8 M H2SO4 to the flasks before diluting them to the 

mark with DI water.

7.9 Spike Solutions

7.9.1 Nitrogen stock spike solution 

(1 mL = 0.50 mg-N): Dissolve 0.955 g ammonium 

chloride (NH4Cl, FW=53.49) in about 400 mL of DI 

water in a 500-mL volumetric flask. Dilute this 

solution to the mark with DI water and mix it 

thoroughly by manual inversion and shaking. Transfer 

the stock spike solution to a 500-mL PyrexTM media 

bottle in which it is stable for 6 months at 4°C.

7.9.2 Phosphorus stock spike solution

(1 mL = 0.20 mg-P): Dissolve 0.439 g potassium 

dihydrogen phosphate (KH2PO4, FW=136.1) in about 

400 mL of DI water in a 500-mL volumetric flask. 

Dilute this solution to the mark with DI water and mix 

it thoroughly by manual inversion and shaking. 

Transfer the stock spike solution to a 500-mL PyrexTM 

media bottle in which it is stable for 6 months at 4°C.

7.9.3 Mixed spike solution (100 μL = 0.005 

mg-N and 0.002 mg-P): Dispense 1 mL each of 

ammonium chloride and orthophosphate stock spike 

solutions into a 10-mL volumetric flask and dilute to 

the mark with DI water. Transfer the mixed spike 

solution to a 15-mL, screw-cap polyethylene centrifuge 

tube in which it is stable for 2 weeks at 4°C.

NOTE: An equivalent mixed spike solution can be 

prepared more conveniently from stock calibrants 

(sections 7.1 and 7.2) by diluting 500 μL of each to 

25 mL in a volumetric flask.

7.10 Digest-Check Stock Solutions

7.10.1 Glycine digest-check stock solution

(1 mL = 1.0 mg-N): Dissolve 3.98 g glycine 

(C2H5NO2•HCl, FW=111.5) in about 400 mL of DI 

water in a 500-mL volumetric flask. Dilute this 

solution to the mark with DI water and mix it 

thoroughly by manual inversion and shaking. Transfer 

the stock digest-check solution to a 500-mL PyrexTM 

media bottle in which it is stable for 6 months at 4°C.

7.10.2 Glycerophosphate digest-check 

stock solution (1 mL = 0.4 mg-P): Dissolve 1.976 g 

glycerophosphate (C3H7O6PNa2•5H2O, FW=306.1) in 

about 400 mL of DI water in a 500-mL volumetric 

flask. Dilute this solution to the mark with DI water 

and mix it thoroughly by manual inversion and 

shaking. Transfer the stock digest-check solution to a 

500-mL PyrexTM media bottle in which it is stable for 6 

months at 4°C.

7.10.3 Glucose digest-check stock solution

(1 mL = 1.25 mg-C): Dissolve 1.564 g glucose 

(C6H12O6, FW=180.2) in about 400 mL of DI water in 

a 500-mL volumetric flask. Dilute this solution to the 

mark with DI water and mix it thoroughly by manual 

inversion and shaking. Transfer the stock digest-check 

solution to a 500-mL PyrexTM media bottle in which it 

is stable for 6 months at 4°C.

Table 5. Volumes of Environmental Resource Associates (ERA) DemandTM nutrient 

concentrate used to prepare 1-liter volumes of check standards used in this study 

 	
			

-N/L, milligrams nitrogen per liter; mg-P/L, milligrams phosphorus per liter] 

Check 

standard 

identity 

ERA 

DemandTM 

volume (μL) 

Volume 

1.8 M H2

SO4 1

(mL) 

Nominal 

concentration 

(mg–N/L) 

Nominal 

concentration 

(mg–P/L) 

Low 100 10.0 0.22 0.11 

High 500 10.0 1.09 0.54 

Very high 1,000 10.0 2.20 1.08 

1

Add H2SO4 only to acidified check standards as described in section 7.8. 
14 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

7.10.4 Mixed digest-check solution (for 

FCC samples—nominal concentration 4 mg-N/L, 1.6 

mg-P/L, and 50 mg-C/L): Dispense 1 mL each of 

glycine and glycerophosphate stock digest-check 

solutions and 10 mL of the glucose digest-check stock 

solution into a 250-mL volumetric flask that contains 

about 200 mL of DI water. Dilute the contents of the 

flask to the mark with DI water and mix it thoroughly 

by manual inversion and shaking. Transfer the stock 

digest-check solution to a 250-mL PyrexTM media 

bottle in which it is stable for 1 month at 4°C.

7.10.5 Acidified mixed digest-check 

solution (for FCA and WCA samples): Prepare this 

digest-check solution identically to the one described 

in section 7.10.4, except add 2.5 mL of 1.8 M H2SO4 to 

the flask before diluting its contents to the mark with 

DI water. Transfer the acidified mixed digest-check 

solution to a 250-mL PyrexTM media bottle in which it 

is stable at 4°C for 1 month.

8. Sample Preparation

8.1 Alkaline persulfate digests are prepared by 

dispensing samples and digestion reagent into 30-mL, 

screw-cap, PyrexTM culture tubes in the volume ratio of 

2 + 1. For filtered samples (FCC bottle types) that were 

prepared robotically, 9.5-mL volumes of samples, 

blanks, calibrants, and reference materials were dosed 

with 4.75-mL volumes of alkaline persulfate digestion 

reagent (see section 6.1.3). This is the maximum 

sample volume that could be delivered by the robotic 

dispenser/diluter system's 10.000-mL syringes because 

0.500 mL of their capacity is expended in the creation 

of air gaps that minimize interaction between samples 

and the DI water carrier fluid. Whole-water samples 

(WCA bottle types) that require vigorous shaking (and 

in a few cases, continuous magnetic stirring) just prior 

to dispensing operations were prepared manually with 

conventional, high-precision, hand-held electronic 

pipets (Rainin EDP PlusTM). Here dispensed volumes 

of sample and digestion reagent (see section 6.1.4) 

were 10.000 and 5.000 mL, respectively. After robotic 

or manual sample and reagent-dispensing operations 

are complete, 100 μL of mixed spike solution (see 

section 7.9.3) is added manually to the designated tube. 

Then all tubes are capped tightly and mixed thoroughly 

either by manual inversion (three times) or with a 

vortex mixer (3, 5-second cycles). The capped tubes 

positioned in a purpose-built, 80-position stainlesssteel rack then are placed in an autoclave where they 

are digested at 121oC and 117.2 kPa for 1 hour. Table 

6 lists the rack protocol suggested for a batch of 80 

tubes consisting of up to 64 samples plus six calibrants, 

four blanks, three quality-control (QC) check solutions, 

one digest-check solution, one duplicate sample, and 

one spiked sample. A step-by-step procedure for 

Table 6. Suggested rack protocol for alkaline persulfate digest preparation 

[ID, identification; QC, quality control; yyyy, year; ddd, Julian day] 

Tube 

number ID Tube number ID Tube number ID Tube number ID 

1 C1 21 yyyyddd0007 41 yyyyddd0027 61 yyyyddd0047 

2 C2 22 yyyyddd0008 42 yyyyddd0028 62 yyyyddd0048 

3 C3 23 yyyyddd0009 43 yyyyddd0029 63 yyyyddd0049 

4 C4 24 yyyyddd0010 44 yyyyddd0030 64 yyyyddd0050 

5 C5 25 yyyyddd0011 45 yyyyddd0031 65 yyyyddd0051 

6 C6 26 yyyyddd0012 46 yyyyddd0032 66 yyyyddd0052 

7 C7 (blank) 27 yyyyddd0013 47 yyyyddd0033 67 yyyyddd0053 

8 blank 28 yyyyddd0014 48 yyyyddd0034 68 yyyyddd0054 

9 blank 29 yyyyddd0015 49 yyyyddd0035 69 yyyyddd0055 

10 blank 30 yyyyddd0016 50 yyyyddd0036 70 yyyyddd0056 

11 QC low 31 yyyyddd0017 51 yyyyddd0037 71 yyyyddd0057 

12 Digest check 32 yyyyddd0018 52 yyyyddd0038 72 yyyyddd0058 

13 QC high 33 yyyyddd0019 53 yyyyddd0039 73 yyyyddd0059 

14 QC very high 34 yyyyddd0020 54 yyyyddd0040 74 yyyyddd0060 

15 yyyyddd0001 35 yyyyddd0021 55 yyyyddd0041 75 yyyyddd0061 

16 yyyyddd0002 36 yyyyddd0022 56 yyyyddd0042 76 yyyyddd0062 

17 yyyyddd0003 37 yyyyddd0023 57 yyyyddd0043 77 yyyyddd0063 

18 yyyyddd0004 38 yyyyddd0024 58 yyyyddd0044 78 yyyyddd0064 

19 yyyyddd0005 39 yyyyddd0025 59 yyyyddd0045 79 Duplicate 

20 yyyyddd0006 40 yyyyddd0026 60 yyyyddd0046 80 Spike 
Analytical Method 15

alkaline persulfate digest preparation is provided in 

NWQL SOP IM0384.0 (available on request).

NOTE: When samples contain large quantities of 

suspended solids, continuous stirring during sample 

aspiration might provide the only means of obtaining 

representative aliquots.

8.2 When the digestion cycle is complete and 

pressure and temperature gages on the autoclave 

indicate 0 kPa and less than 80°C, remove the alkaline 

persulfate digests from the autoclave and allow them to 

cool sufficiently to be handled comfortably. Then mix 

the contents of each capped digestion tube by manual 

inversion (three times) or with a vortex mixer (three, 

5-second cycles). FCC and FCA digests can be poured 

into analyzer cups immediately after mixing. Wait 

about 1 hour after mixing WCA digests to allow 

suspended solids to settle. If it is not possible to decant 

or pipet a clear supernatant solution from digest tubes 

into analyzer cups, then suspended solids must be 

removed by 0.45-μm filtration prior to colorimetric 

analysis. Note that tightly capped digests can be stored 

at room temperature for several days (4 days was the 

maximum delay tested) before their nitrogen and 

phosphorus concentrations are determined by 

automated colorimetry.

9. Instrument Performance

An 80-tube batch of samples, calibrants, and 

reference materials can be prepared robotically and 

made ready for digestion in about 1 hour. Digestion 

time—including warm up, cool down, and 

postdigestion mixing—is about 2 hours. The NWQL 

Nutrients Unit has two autoclaves, each of which can 

hold two, 80-tube racks of alkaline persulfate digests. 

Nitrate and orthophosphate in alkaline persulfate 

digests can be determined simultaneously with the 2-

channel air-segmented continuous flow analyzer at a 

rate of about 100 samples per hour with less than 1 

percent interaction. Thus, using a combination of 

robotic and manual sample preparation, up to six racks 

(384 actual samples out of 480 total tubes) of alkaline 

persulfate digests can be prepared in an 8-hour day. 

This estimate assumes the use of both NWQL 

autoclaves and a combination of robotic (FCC 

samples) and manual (WCA samples) sample 

preparation. Likewise, up to six racks of previously 

digested samples can be analyzed for nitrate and 

orthophosphate in an 8-hour day. This production rate 

assumes that digest analysis can lag sample digestion 

by 1 to 3 days.

10. Calibration

With a second-order polynomial least-squares 

curve-fitting function (y = a+bx+cx2, where y is the 

baseline and blank-corrected peak height and x is the 

nominal concentration), calibration plots with 

correlation coefficients (r2

) greater than 0.999 are 

achieved routinely. Typical calibration plots for nitrate 

and orthophosphate in alkaline persulfate digests are 

shown in figures 3 and 4.

NOTE: In addition to baseline drift correction, a 

digestion blank correction must be applied to 

calibrants, check standards, and samples prior to 

calculation of final results, as described in sections 

12.3 and 12.4.

11. Procedure and Data Evaluation

Set up the continuous flow analyzer analytical 

cartridges as shown in figures 1 and 2. Turn on 

electrical power to all system modules and put fresh 

sampler wash reservoir solution and reagents on-line. 

After about 10 minutes, verify that the sample and 

reference outputs of both photometers are set at about 

5 volts. A suggested sampler tray protocol for 

automated determination of nitrate and orthophosphate 

in alkaline persulfate digests is listed in table 7.

NOTE: To minimize errors that result from 

contaminated analyzer cups, rinse them several times 

with the solution they are to contain before placing 

them on the analyzer sampler tray.

NOTE: The full-scale absorbance range control (STD 

CAL) of photometers should not require daily 

adjustment. Between-analysis/between-day variations 

in baseline-absorbance level and calibration curve 

slope of about ±5 percent are acceptable. Adjustment 

of the STD CAL control to compensate for larger 

variations in sensitivity or baseline (reagent blank) 

levels will only mask underlying problems, such as 

incipient light source failure, partially clogged flow 

cells, or contaminated or improperly prepared reagents, 

any of which could compromise analytical results.
16 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

0.00 1.25 2.50 3.75 5.00

0.00

1.00

2.00

3.00

4.00

ABSORBANCE, IN VOLTS

NITROGEN CONCENTRATION, IN MILLIGRAMS PER LITER

 mg-N/L Volts 

 5.00 4.322

 3.75 3.272

 2.50 2.175

 1.25 1.122

 0.50 0.469

 0.10 0.091

 0.00 0.000

y = a + bx + cx2

Parameter Value Error r2

 a 0.010 0.010 0.99994

 b 0.886 0.013

 c -0.005 0.003

0.0 0.5 1.0 1.5 2.0

0.00

1.00

2.00

3.00

4.00

PHOSPHORUS CONCENTRATION, IN MILLIGRAMS PER LITER

ABSORBANCE, IN VOLTS

y = a + bx + cx2

Parameter Value Error r2

 a 0.005 0.006 0.99998

 b 2.132 0.018

 c 0.024 0.009

 mg-P/L Volts 

 2.00 4.368

 1.50 3.256

 1.00 2.154

 0.50 1.083

 0.20 0.446

 0.04 0.084

 0.00 0.000

Figure 4. Typical calibration graph for total phosphorus determined as 

orthophosphate in alkaline persulfate digests.

Figure 3. Typical calibration graph for total nitrogen determined as 

nitrate in alkaline persulfate digests.
Analytical Method 17

12. Calculations

12.1 Instrument calibration requires preparing a 

set of solutions (calibrants) in which the analyte 

concentration is known. These calibrants are digested 

along with samples and used to establish a calibration 

function that is estimated from a least-squares fit of 

nominal calibrant concentrations (x) in relation to peak 

absorbance (y). A second-order polynomial function 

(y = a+bx+cx2) usually provides improved 

concentration estimates at the upper end of the 

calibration range than a more conventional linear 

function (y = a+bx). Accuracy is not lost when a 

second-order fit is used, even if the calibration function 

is strictly linear, because, in this case, the value 

estimated for the quadratic parameter c will approach 

zero.

12.2 Before the calibration function can be 

estimated, the baseline absorbance component of 

measured peak heights, including drift (continuous 

increase or decrease in the baseline absorbance during 

the course of an analysis), if present, needs to be 

removed. Baseline absorbance in continuous flow 

analysis is analogous to the reagent blank absorbance 

in batch analysis. Correction for baseline absorbance is 

an automatic function of most data acquisition and 

processing software sold by vendors of continuous 

flow analyzers.

NOTE: These correction algorithms are based on 

linear interpolation between initial and intermediate or 

final baseline measurements, and so they do not 

accurately correct for abrupt, step-changes in baseline 

absorbance that usually indicate partial flow-cell 

blockage. It is prudent, therefore, to reestablish 

baseline absorbance at intervals of 20 samples or so.

Table 7. Suggested analyzer sample tray protocol for automated determination of nitrate and orthophosphate in 

alkaline persulfate digests

[#, number; ID, identification; SYNC, synchronization peak; CO, carry-over peak; W, wash; UB, undigested blank; 

DB, digested blank; CCV, continuing calibration verification; QC, quality control; yyyy, year; ddd, Julian day] 

Cup # ID Cup # ID Cup # ID Cup # ID 

1 SYNC 24 yyyyddd0006 47 yyyyddd0029 70 yyyyddd0050 

2 CO (C6) 25 yyyyddd0007 48 yyyyddd0030 71 yyyyddd0051 

3 (C6) 26 yyyyddd0008 49 yyyyddd0031 72 yyyyddd0052 

4 W 27 yyyyddd0009 50 yyyyddd0032 73 yyyyddd0053 

5 C1 28 yyyyddd0010 51 UB 74 yyyyddd0054 

6 C2 29 yyyyddd0011 52 W (DB) 75 yyyyddd0055 

7 C3 30 yyyyddd0012 53 yyyyddd0033 76 yyyyddd0056 

8 C4 31 yyyyddd0013 54 yyyyddd0034 77 yyyyddd0057 

9 C5 32 yyyyddd0014 55 yyyyddd0035 78 yyyyddd0058 

10 C6 33 yyyyddd0015 56 yyyyddd0036 79 yyyyddd0059 

11 C7 34 yyyyddd0016 57 yyyyddd0037 80 yyyyddd0060 

12 W 35 yyyyddd0017 58 yyyyddd0038 81 yyyyddd0061 

13 CCV 36 yyyyddd0018 59 yyyyddd0039 82 yyyyddd0062 

14 UB1 37 yyyyddd0019 60 yyyyddd0040 83 yyyyddd0063 

15 QC low2 38 yyyyddd0020 61 yyyyddd0041 84 yyyyddd0064 

16 Digest check3 39 yyyyddd0021 62 yyyyddd0042 85 duplicate 

17 QC high2

 40 yyyyddd0022 63 yyyyddd0043 86 Spike 

18 QC very high2 41 yyyyddd0023 64 yyyyddd0044 87 UB 

19 yyyyddd0001 42 yyyyddd0024 65 yyyyddd0045 88 CCV 

20 yyyyddd0002 43 yyyyddd0025 66 yyyyddd0046 89 UB 

21 yyyyddd0003 44 yyyyddd0026 67 yyyyddd0047 90 W (DB) 

22 yyyyddd0004 45 yyyyddd0027 68 yyyyddd0048 

23 yyyyddd0005 46 yyyyddd0028 69 yyyyddd0049 

1

Undigested blank (sampler wash reservoir solution, see section 6.2.1). 

2

NWQL Check Standard, see sections 7.7 and 7.8. 

3

Digest-check sample; see sections 7.10.4 and 7.10.5. 
18 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

12.3 After peaks are baseline corrected, they need 

to be digestion-blank corrected.

This correction can be applied in several ways:

1. Subtract the baseline-corrected absorbance of 

the digestion blank—compute an average 

concentration if multiple digested blanks are 

included in each block—from the baselinecorrected absorbance of all calibrants, check 

standard, and samples in the block. Then 

estimate regression parameters (a, b, and c 

terms) for the calibration function by using a 

second-order polynomial least-squares 

algorithm. For second and higher order 

calibration functions, use the NewtonRaphson successive approximations 

algorithm (Draper and Smith, 1966; Swartz, 

1976, 1977, 1979) to convert corrected peak 

heights into concentrations. 

2. Designate digestion blanks as a calibrant with a 

nominal concentration of zero. In this case 

the resulting calibration function will have a 

positive y-intercept that approximates the 

baseline-corrected absorbance of the 

digestion blank. If this method is used, be 

sure that the curve-fitting algorithm does not 

force a zero y-intercept by including one or 

more “dummy” (0,0) points in the data set 

used for calibration.

3. Designate digested blanks as baseline correction 

samples—that is, “W” in the FasPacTM 

software used to acquire and process data at 

the NWQL. In this case initial, intermediate 

(if included), and final baselines are 

interpolated between digested blank peak 

maxima. Thus, baseline and digestion blanks 

are corrected in a single operation.

NOTE: Digestion blanks were corrected for data in 

this report by using method 3. However, analytical 

results calculated by the other two methods should be 

equivalent. Regardless of the blank correction 

algorithm chosen, make sure that it is documented in 

the SOP and that analysts understand it. The SOP for 

these methods must be updated whenever any changes 

in data acquisition and processing software or in 

calculation algorithms are implemented.

12.4 Most software packages provide a data base 

for entering appropriate dilution factors. Usually these 

factors can be entered before or after samples are 

analyzed. If dilution factors are entered, reported 

concentrations will be compensated automatically for 

the extent of dilution. The dilution factor is the number 

by which a measured concentration must be multiplied 

to obtain the analyte concentration in the sample prior 

to dilution. For example, dilution factors of 2, 5, and 

10 indicate that sample and diluent were combined in 

proportions of 1+1, 1+4, and 1+9, respectively.

13. Reporting Results

Total nitrogen (lab codes 2754, 2755, 2756)

• 2 decimal places for concentrations up to 5.00 

mg-N/L

• 2 significant figures for concentrations greater 

than 5.00 mg-N/L

Total phosphorus (lab codes 2757, 2758, 2759)

• 2 decimal places for concentrations up to 2.00 

mg-P/L

• 2 significant figures for concentrations greater 

than 2.00 mg-P/L

14. Detection Levels, Bias, and Precision

14.1 Method detection limits (MDL) for 

composited, low-concentration FCC and WCA 

samples (five of each) were estimated using the U.S 

Environmental Protection Agency (1997) 

protocol—see table 8. Target concentrations for 

nitrogen and phosphorus in FCC and WCA composite 

samples were 0.05 mg-N/L and 0.02 mg-P/L, 

respectively. The MDL for nitrogen was 0.015 

mg-N/L and for phosphorus was 0.007 mg-P/L. 

Laboratory reporting levels (LRL) will be about twice 

the MDL concentrations.

14.2 Table 9 lists the average and standard 

deviation of 9987L, 9987H, and 9987VH QC check 

solutions that were included in every rack of alkaline 

persulfate digests. Most probable values (MPVs) and 

standard deviations in table 9 were published by the 

USGS Branch of Quality Systems for the 2002 water 

year (12-month period ending September 30 each year 

is called the “water year”). In all cases, total nitrogen 

and total phosphorus concentrations determined for 

these reference materials by the alkaline persulfate 

digestion method were tightly centered around 

published MPVs and well within published control 

limits.
Analytical Method 19

Table 8. Data and calculations used to estimate method detection limits (MDL) for nitrogen and phosphorus 

in unacidified (FCC) and acidified (WCA) samples following alkaline persulfate digestion. Low-concentration 

FCC and WCA samples (five of each) were composited for these determinations 

[mg-N (-P)/L, milligrams nitrogen (or phosphorus) per liter; %, percent; MDL, method detection limit] 

Concentration found (mg-N/L or mg-P/L) Target 

concentration 

[mg-N (-P)/L] 

Dissolved 

nitrogen 

(unacidified) 

Total 

nitrogen 

(acidified) 

Dissolved 

phosphorus 

(unacidified) 

Total 

phosphorus 

(acidified) 

0.05 (0.02) 0.064 0.041 0.026 0.033 

0.05 (0.02) .078 .042 .024 .029 

0.05 (0.02) .072 .035 .026 .029 

0.05 (0.02) .066 .035 .029 .027 

0.05 (0.02) .067 .032 .026 .029 

0.05 (0.02) .066 .039 .023 .027 

0.05 (0.02) .071 .026 .022 .026 

0.05 (0.02) .063 .035 .026 .026 

Average .068 .035 .025 .028 

Standard deviation 

 

.005 

 

.005 

 

.002 

 

.002 

Number of values 8 8 8 8 

Degrees of freedom 7 7 7 7 

t-value (1-sided, 99%) 2.998 2.998 2.998 2.998 

MDL .015 .015 .007 .007 

Table 9. Most probable values and standard deviations for reference samples 9987L, 9987H, and 9987VH 

along with averages and standard deviations of these reference materials that were included in every rack of 

alkaline persulfate digests 

[ID, identification of reference sample; MPV, most probable value; FCC, filtered, chilled (bottle type); WCA, whole 

water, chilled, acidified (bottle type); mg-N/L, milligrams nitrogen per liter; mg-P/L, milligrams phosphorus per liter; 

±, plus or minus] 

 High-flow samples Low-flow samples ID MPV FCC1

WCA2FCC3WCA4

Alkaline persulfate dissolved and total nitrogen concentration (mg-N/L) 

9987L 0.22 ± 0.08 0.21 ± 0.03 0.21 ± 0.03 0.19 ± 0.03 0.20 ± 0.02 

9987H 1.09 ± 0.15 1.09 ± 0.03 1.09 ± 0.03 1.06 ± 0.08 1.04 ± 0.04 

9987VH 2.20 ± 0.24 2.27 ± 0.05 2.18 ± 0.06 2.16 ± 0.07 2.13 ± 0.06 

Alkaline persulfate dissolved and total phosphorus concentration (mg-P/L) 

9987L 0.108 ± 0.008 0.105 ± 0.004 0.104 ± 0.004 0.107 ± 0.006 0.105 ± 0.004 

9987H 0.54 ± 0.02 0.54 ± 0.01 0.55 ± 0.02 0.57 ± 0.02 0.54 ± 0.01 

9987VH 1.08 ± 0.05 1.13 ± 0.02 1.10 ± 0.03 1.13 ± 0.03 1.09 ± 0.02 

1

Number of points: n = 19; 2

n = 21; 3n = 21; 4n = 18. 
20 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

14.3 Spike Recoveries

Median, 90th and 10th percentiles of percent spike 

recoveries measured in samples collected during highflow and low-flow conditions are listed in table 10. 

Median spike recoveries for nitrogen (0.5 mg-N/L as 

glycine) ranged from about 92 to100 percent and for 

phosphorus (0.2 mg-P/L as glycerophosphate) from 

about 86 to 108 percent.

14.4 Duplication of Results

Median, tenth percentiles, and ninetieth percentiles 

for concentration differences for duplicate samples 

collected during the nominally high- and low-flow 

conditions are listed in table 11. Median concentration 

differences between duplicate analyses are about the 

same as the MDLs. Larger tenth-percentile differences 

for whole-water samples that were collected during 

nominally high-flow conditions in relation to those of 

filtered water samples likely reflect the difficulty of 

obtaining reproducible aliquots from samples that 

contain large amounts of suspended solids. Such 

samples were purposely chosen as duplicates to assess 

“worst-case” digest-preparation sampling precision.

ANALYTICAL PERFORMANCE AND 

COMPARATIVE RESULTS

This section documents analytical performance of 

the alkaline persulfate digestion method (I-2650-03/

4650-03) developed and adapted for use at the NWQL 

as an alternative to USGS Kjeldahl digestion methods 

for nitrogen (I-2515-91/4515-91) and phosphorus 

(I-2610-91/4610-91). It also provides statistical and 

graphical analysis of data and interpretation of results 

for about 2,100 dissolved and whole-water samples 

that were collected during nominally high- and lowflow conditions and analyzed by alkaline persulfate 

and Kjeldahl digestion methods.

Table 10. Spike recoveries of glycine and glycerophosphate in randomly selected high-flow and low-flow 

samples that were included in every rack of alkaline persulfate digests 

[n, number of samples; DNAlkP, alkaline persulfate dissolved nitrogen; TNAlkP, alkaline persulfate total 

nitrogen; DPAlkP, alkaline persulfate dissolved phosphorus; TPAlkP, alkaline persulfate total phosphorus] 

 High-flow samples Low-flow samples 

 Percent recovery Percent recovery n Median 90th 10th n Median 90th 10th 

DNAlkP 18 100.3 108.6 90.1 18 95.0 103.2 88.7 

TNAlkP 22 95.1 103.1 84.0 18 92.1 101.7 83.0 

DPAlkP 18 97.9 112.9 86.5 17 108.3 119.2 93.4 

TPAlkP 22 85.8 93.3 69.5 18 99.6 107.5 91.4 

Table 11. Concentration differences between selected samples prepared and analyzed in duplicate 

in each block of alkaline persulfate digests 

[n, number of samples; mg-N/L, milligrams nitrogen per liter; mg-P/L, milligrams phosphorus per liter; 

DNAlkP, alkaline persulfate dissolved nitrogen; TNAlkP, alkaline persulfate total nitrogen; DPAlkP, alkaline 

persulfate dissolved phosphorus; TPAlkP, alkaline persulfate total phosphorus] 

High-flow samples Low-flow samples 

Concentration difference 

for mg-N/L or mg-P/L 

(percentile) 

 Concentration difference 

for mg-N/L or mg-P/L 

(percentile) n

Median 90th 10th 

n

Median 90th 10th 

DNAlkP 20 0.011 0.050 -0.023 20 -0.023 0.008 -0.109 

TNAlkP 20 -0.007 0.052 -0.296 20 -0.024 0.027 -0.093 

DPAlkP 20 0.000 0.009 -0.024 20 -0.002 0.010 -0.034 

TPAlkP 20 0.000 0.015 -0.040 20 -0.004 0.006 -0.023 
Analytical Performance and Comparative Results 21

Analytical Performance

Prior to beginning the large-scale evaluation and 

validation study with samples collected during 

nominally high- and low-flow conditions, preliminary 

experiments were performed to establish performance 

of the alkaline persulfate digestion method. Recoveries 

of nitrogen and phosphorus from individual nitrogenand phosphorus-containing compounds that were 

prepared in deionized water are listed in tables 12 

and 13. Inspection of table 12 reveals greater than 95-

percent recovery of nitrogen for compounds tested. 

The somewhat lower recoveries obtained for 

phosphorus compounds listed in table 13 result from 

the lower purity of test compounds (phenyl phosphate 

and phytic acid ≤95 percent according to vendor labels; 

ATP was purchased and first opened in 1991). 

Comparable, though slightly higher, phosphorus 

recoveries for these compounds by acid persulfate 

digestion (USEPA method 365.1—the generally 

accepted reference method for total phosphorus 

determinations), which also are shown in table 13, 

substantiate this assertion. Other researchers (Ebina 

and others, 1983; Hosomi and Sudo, 1986; Ameel and 

others, 1993), who used alkaline persulfate digestion 

methods similar to the one developed at the NWQL, 

reported phosphorus recoveries greater than 95 percent 

for a variety of phosphorus-containing compounds, 

including ATP.

Results from an experiment to assess nitrogen 

recovery in the presence of organic carbon (OC) are 

shown in figure 5. In these experiments a series of 

solutions containing 2.5 mg NH4

+-N/L and increasing 

concentrations of OC (as glucose) were digested and 

analyzed for nitrogen. Data plotted in figure 5 indicate 

that complete oxidation of ammonium to nitrate was 

achieved for OC concentrations up to 150 mg/L. 

Similar results have been reported previously (Langer 

and Hendrix, 1982; Cabrera and Beare, 1993). OC in 

surface- and ground-water samples analyzed at the 

NWQL rarely exceeds 150 mg/L. Note, however, that 

nitrogen recovery in Kjeldahl digests is quantitative at 

OC concentrations 10 to 20 times greater than the 150-

mg/L limit typical for alkaline persulfate digestion 

methods (Ebina and others, 1983).

During preliminary validation work, the cause of 

low nitrogen recovery in about 10 WCA samples was 

traced to overacidification at collection sites. When 

these samples were dosed with alkaline persulfate 

reagent, the resulting pH was less than 7. As discussed 

previously in section 2.2, an initial pH greater than 12 

is necessary for complete oxidation of ammonium and 

organic nitrogen to nitrate. For this reason the pH of all 

WCA samples was checked with narrow range 

colorimetric test strips during the large-scale 

evaluation and validation study. Nitrogen 

Table 12. Recovery of inorganic and organic nitrogen 

from representative compounds 

[mg-N/L, milligrams nitrogen per liter; ±, plus or minus] 

Nitrogen 

compound 

Nominal 

concentration 

(mg-N/L) 

Found 

(mg-N/L) 

Recovery

(percent) 

Ammonia 2.5 2.51 ± 0.05 100.5 

Urea 2.5 2.50 ± 0.06 100.0 

Nicotinic acid 2.5 2.47 ± 0.04 98.7 

Glycine 2.5 2.50 ± 0.07 97.9 

Table 13 Recovery of organic phosphorus from representative compounds by alkaline persulfate and lowlevel acid persulfate digestion methods 

[mg-P/L, milligrams phosphorus per liter; ±, plus or minus] 

Alkaline persulfate 

method Acid persulfate method 

Compound 

Nominal 

concentration 

(mg-P/L) 

Found 

(mg-P/L) 

Recovery 

(percent) 

Found 

(mg-P/L) 

Recovery 

(percent) 

Adenosine tri-phosphate (ATP) 0.200 0.166 ± 0.000 83.0 0.176 ± 0.001 88.0 

1.000 0.87 ± 0.03 86.8 

Glycerophosphate 0.200 0.196 ± 0.007 100.2 0.204 ± 0.002 102.1 

1.000 1.019 ± 0.008 101.9 

Phenyl Phosphate 0.200 0.168 ± 0.004 84.2 0.179 ± 0.000 89.5 

1.000 0.872 ± 0.002 87.2 

Phytic Acid 0.200 0.177 ± 0.002 88.4 0.180 ± 0.001 91.8 

1.000 0.906 ± 0.009 90.6 
22 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

concentration results for WCA samples with pH 

outside the expected range of 1.6 to 1.9 were 

disqualified. For more information, see section 3.1.4. 

About 150 results for samples with medium codes 

other than 6 (ground water) and 9 (surface 

water)—specifically, Q (quality-assurance sample, 

artificial), R (quality-assurance sample, surface water), 

S (quality-assurance sample, ground water), 2 

(leachate), and 5 (elutriation)—also were not included 

in graphical and statistical analyses.

In Kjeldahl digestion procedures, digests are 

evaporated to near dryness and then resolvated with DI 

water prior to analytical determinations. Variation in 

the postdigestion volume of DI water added to each 

tube—and therefore in estimated mass-per-unit volume 

nitrogen and phosphorus concentrations in resolvated 

digests—is a function of the DI water dispenser 

precision, typically 2 to 3 percent. Alkaline persulfate 

digests, in contrast, are tightly capped and lose little 

water during digestion. After digestion, nitrogen and 

phosphorus are determined directly without volume 

adjustment. It was of interest, therefore, to assess the 

variation in liquid loss during alkaline persulfate 

digestion. To this end, pre- and postdigestion weights 

for one, 80-tube batch of prepared alkaline persulfate 

digests were measured to the nearest 0.01 g and 

recorded. A weight of 15 g (10 mL of sample + 5 mL 

of digestion reagent) was assumed in percent weightloss calculations using equation 1 below. The results 

from this experiment, which indicate a weight loss of 3 

percent or less for 85 percent of all tubes, are shown in 

figure 6. The maximum percent weight loss observed 

was 6 percent.

Comparative Results for Nitrogen

In discussions that follow, the designations KDN 

and KTN apply to Kjeldahl digestion dissolved 

nitrogen (ammonium + organic nitrogen determined in 

filtered-water digests) and Kjeldahl digestion total 

nitrogen (ammonium + organic nitrogen determined in 

acidified, whole-water digests), respectively. When 

filtered- and whole-water samples are considered 

0 150 300 450 600

0

20

40

60

80

100

AMMONIUM RECOVERY, IN PERCENT

GLUCOSE CONCENTRATION, IN MILLIGRAMS CARBON PER LITER

Figure 5. Percent recovery of nitrogen from a series of 2.5 mg NH4

+-N solutions that 

contained increasing concentrations of organic carbon (OC) as glucose. See text for 

additional details.

(1) Percent weight loss = Digest weightinitial Digest weightfinal –

15g ------------------------------------------------------------------------------------------------ × 100
Analytical Performance and Comparative Results 23

together, the designation Kjeldahl nitrogen (KN) is 

applied. The designations alkaline persulfate digestion 

dissolved nitrogen (DNAlkP), alkaline persulfate 

digestion total nitrogen (TNAlkP), and alkaline 

persulfate nitrogen (NAlkP) are applied analogously. 

Note also that nitrate-corrected DNAlkP , TNAlkP , and 

NAlkP concentrations are those from which nitrate + 

nitrite concentrations have been subtracted to make 

them operationally equivalent to KDN, KTN, and KN 

concentrations.

A logarithmic scatter plot of nitrate-corrected NAlkP

concentrations and KN concentrations around a unity 

slope line—that is, the line of equal relation—for 

paired data combined from the large-scale validation 

experiments is shown in figure 7. Despite the large 

scatter between individual data pairs, linear regression 

analysis of these data indicate good correlation 

between nitrate-corrected NAlkP and KN concentrations—KN = (1.023 ± 0.003) NAlkP + 0.038 ± 0.004, 

r

2

 = 0.976. The positive y-intercept and slightly greater 

than unity slope of the regression line indicate low bias 

for nitrate-corrected NAlkP in relation to KN, which 

might be interpreted as low nitrogen recovery for the 

alkaline persulfate digestion method. An alternate 

interpretation—that KN concentrations are biased high 

because a small fraction of nitrate present in samples is 

reduced to ammonium during Kjeldahl digestion—also 

could account for observed concentration differences. 

Interference by nitrate during Kjeldahl digestion 

(American Public Health Association, 1998c, p. 4-123; 

Patton and Truitt, 2000) is well known.

To explore this alternative interpretation further, 

differences between nitrate-corrected NAlkP and KN 

concentrations (y-axes) were plotted as a function of 

nitrate concentrations (x-axes) in the four panels of 

figure 8. In this figure panels A and B relate to data for 

filtered surface- and ground-water samples; panels C 

and D relate to data for whole-water acidified surfaceand ground-water samples. Nitrate concentrations are 

plotted on logarithmic scales to provide equal linear 

spacing for each decade of nitrate concentration. Lines 

of zero concentration difference were added to 

facilitate visual interpretation of data. With the 

exception of some unexplained outliers, differences 

between nitrate-corrected DNAlkP and KDN 

concentrations in filtered samples (fig. 8A and 8B) tend 

to scatter symmetrically about the lines of zero 

difference up to nitrate concentrations of about 

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

0

5

10

15

20

25

30

35

NUMBER OF TUBES

POSTDIGESTION WEIGHT LOSS, IN PERCENT

0

10

20

30

40

50

60

70

80

90

100

 PERCENT OF ALL TUBES

Figure 6. Histogram of postdigestion weight loss expressed as a percent of initial digest weight for one batch of 

80 alkaline persulfate digests. Weights of capped digest tubes, each containing precisely dispensed volumes of 

sample and reagent, were weighed before and after digestion. Left and right y-axes relate to gray bars and the 

dashed line, respectively. Additional details can be found in supporting text.
24 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

1 mg-N/L. At higher nitrate concentrations, 

differences between nitrate-corrected DNAlkP and KDN 

concentrations increase about the line of zero 

difference—erratically for filtered ground water and 

negatively for filtered surface water. In contrast, 

concentration differences between nitrate-corrected 

TNAlkP and KTN for unfiltered, acidified samples (fig. 

8C and 8D) are predominately negative with 

differences becoming more negative as nitrate 

concentrations increase. This trend is particularly 

evident for unfiltered, acidified surface water.

The result of sorting data from each panel in figure 

8 according to nominal flow conditions at the time of 

sample collection and recasting them as box plots is 

shown in figure 9. In this figure, concentration 

differences between nitrate-corrected NAlkP and KN for 

samples collected during nominally high-flow (HF) 

and low-flow (LF) conditions are grouped into three 

nitrate concentration ranges: NO3

-

-N ≤ 0.1 mg/L, 0.1 

mg/L < NO3

-

-N ≤ 1.0 mg/L, and NO3

-

-N > 1.0 mg/L. 

Figure 9 further substantiates the hypothesis that 

concentration differences between nitrate-corrected 

NAlkP and KN likely result from the well known, though 

poorly characterized, high-temperature reactions 

between nitrate and natural organic matter (NOM) that 

can produce positive (reduction of nitrate to 

ammonium) or negative (oxidation of ammonium to 

nitrous oxide) interference in Kjeldahl nitrogen 

determinations (see section 3.1.5 and Patton and Truitt, 

2000). Positive nitrate interference in KN 

concentrations predominates for surface-water samples 

and is greater for whole-water samples than for 

filtered-water samples. This result is consistent with 

typically larger NOM concentrations in whole-water 

samples than in filtered-water samples. Nitrate appears 

to interfere positively and negatively in KN 

concentrations for ground-water samples, although the 

trends are less clear than for surface-water samples. In 

general, differences between nitrate-corrected NAlkP

and KN concentrations were least for samples with 

nitrate concentrations less than 0.1 mg NO3

-

-N/L—a 

finding consistent with nitrate interference during 

Kjeldahl digestion. Complete two-population, paired 

t-test results for subsets of nitrate-corrected DNAlkP and 

0.01 0.1 1 10

0.01

0.1

1

10

Line of equal relation

n = 2066

KJELDAHL NITROGEN, IN

MILLIGRAMS NITROGEN PER LITER

ALKALINE PERSULFATE NITROGEN MINUS

NITRATE, IN MILLIGRAMS NITROGEN PER LITER

Figure 7. Logarithmic scatter plot of nitrate-corrected NAlkP concentrations (x-axis) and KN concentrations (y-axis) 

around the line of equal relation for 2,066 data pairs combined from large-scale validation experiments. The linear 

regression equation for these data is y = (1.023 ± 0.003) x + 0.038 ± 0.004, with a correlation coefficient (r2) of 

0.976.
Analytical Performance and Comparative Results 25

KDN concentrations in filtered-water samples are 

listed in table 14. Nitrate-corrected TNAlkP and KTN 

concentrations in acidified whole-water samples are 

listed in table 15. 

Comparative Results for Phosphorus

A logarithmic scatter plot of PAlkP concentrations 

(x-axis) and KP concentrations (y-axis) around a unity 

slope line for 2,093 data pairs combined from high- and 

low-flow phases of validation experiments is shown in 

figure 10. This plot reveals good correlation among 

phosphorus concentrations determined by the PAlkP and 

KP digestion methods. The slope and y-intercept of the 

linear least squares regression of these data—KP = 

(0.994 ± 0.002) PAlkP + 0.0003 ± 0.0005; correlation 

coefficient (r2

) = 0.994 closely approximate 1 and 0. 

A two-population, paired t-test confirmed the null 

hypothesis that the difference between means of 

phosphorus concentrations for these 2,093 paired 

results determined by the PAlkP and KP digestion 

methods were not significantly different from zero at 

the p = 0.05 level. Differences between means of 

alkaline persulfate phosphorus and Kjeldahl 

phosphorus concentrations for some subsets of these 

data, which were grouped according to water type and 

flow conditions at the time of sample collection, were 

statistically different from zero at the p = 0.05 level. In 

all such cases, however, differences between means 

were less than method detection limits—0.007 mg-P/L 

for PAlkP and 0.02 mg-P/L for KP—and therefore were 

not analytically significant. Complete results for these 

t-tests are listed in table 16.

-1.0

-0.5

0.0

0.5

1.0 A

Filtered Surface W ater (n = 543)

B

Filtered Ground Water (n = 453)

0.01 0.1 1 10 50

-1.0

-0.5

0.0

0.5

1.0 C

(NAlkP - NO3

-

-N) - KN, IN MILLIGRAMS NITROGEN PER LITER

NITRATE CONCENTRATION, IN MILLIGRAMS NITROGEN PER LITER

Unfiltered, acidified surface water (n = 959)

0.01 0.1 1 10 50

D

Unfiltered, acidified ground water (n = 111)

Figure 8. Concentration differences between nitrate-corrected alkaline persulfate 

digestion nitrogen (NAlkP) and Kjeldahl digestion nitrogen (KN) plotted about the line of zero 

difference as a function of nitrate concentration.
26 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

SUMMARY AND CONCLUSIONS

An alkaline persulfate digestion method and 

automated colorimetric finishes for simultaneous 

nitrogen and phosphorus determinations in filtered and 

whole-water acidified water samples were developed 

and validated. This method is more sensitive, accurate, 

and uses less toxic reagents than Kjeldahl digestion 

methods, such as U.S. Geological Survey (USGS) 

I-2515/4515-91 and U.S. Environmental Protection 

Agency (USEPA) 351.2 for nitrogen and USGS I2610/4610-91 and USEPA 365.4 for phosphorus. Data 

in this report result from about 2,100 filtered and 

whole-water samples that were analyzed for alkaline 

persulfate dissolved and total nitrogen (DNAlkP and 

TNAlkP), Kjeldahl dissolved and total nitrogen (KDN 

and KTN), alkaline persulfate dissolved and total 

phosphorus (DPAlkP and TPAlkP), and Kjeldahl dissolved 

and total phosphorus (KDP and KTP). All filtered and 

whole-water samples analyzed by the alkaline 

persulfate digestion method also were analyzed for 

dissolved nitrate + nitrite, ammonium, and 

orthophosphate on the same day that digests were 

prepared. Results of these analyses were compared by 

statistical and graphical methods. About half the data 

HF LF HF LF HF LF -0.30

-0.15

0.00

0.15

0.30

n = 42

n = 64

n = 75

n = 136 n = 131 n = 95

Filtered Surface Water

HF LF HF LF HF LF

n = 71 n = 82 n = 60 n = 60

n = 63

n = 117

Filtered Ground Water

HF LF HF LF HF LF

-0.45

-0.30

-0.15

0.00

0.15

n = 97

n = 115

n = 177

n = 217 n = 237

n = 116

Unfiltered, acidified surface water

HF LF HF LF HF LF

NO3

-

-N≤0.1 0.1<NO3

-

-N≤1.0 NO3

- NO3 -N>1.0 -

-N≤0.1 0.1<NO3

-

-N≤1.0 NO3

-

-N>1.0

(NAlkP - N03

--N) - KN, IN MILLIGRAMS NITROGEN PER LITER

n = 8

n = 5

n = 7

n = 21

n = 48

n = 23

Unfiltered, acidified ground water

Figure 9. Boxplots of concentration differences between nitrate-corrected alkaline persulfate nitrogen (NAlkP) 

and Kjeldahl nitrogen (KN) for surface- and ground-water samples collected during nominally high- and low-flow 

conditions. Data in each panel are grouped according to nominal flow conditions (HF = high-flow; LF = low-flow) 

at the time of sample collection and dissolved nitrate concentrations (milligram nitrogen per liter). In each 

boxplot, open squares, hinges, gates and whiskers indicate average, median, 75th and 25th percentiles, and 

90th and 10th percentiles for differences between nitrate-corrected NAlkP and KN.
Summary and Conclusions 27

Table 14. Results of paired t-tests for nitrate-corrected alkaline persulfate dissolved nitrogen and Kjeldahl dissolved nitrogen concentrations determined in filtered-water 

samples. Distributions of nitrate concentrations for each subgroup are shown in the rightmost three columns

[mg-N/L, milligrams nitrogen per liter; DNAlkP, alkaline persulfate digestion dissolved nitrogen; KDN, Kjeldahl digestion dissolved nitrogen; n, number of samples; max, maximum; 

75th, 75th percentile; <, less than; ≤, less than or equal to; >, greater than]

1pcalc is the probability that population means of nitrate-corrected DNAlkP and KDN concentrations are the same—that is, difference between the population means is statistically 

equivalent to zero—on the basis of calculated paired t-tests. Difference between population means is significant at the 95-percent confidence level (p0.05) when pcalc is less than 0.05 and 

at the 99-percent confidence level (p0.01) when pcalc is less than 0.01.

Mean 

(mg-N/L) Variance (mg-N/L) Significance1 Nitrate (mg-N/L) Filtered ground water DNAlkP KDN Difference (mg–N/L) DNAlkP KDN 

n

pcalc p0.05 p0.01 Median Max 75th 

All 0.340 0.333 0.007 2.387 2.397 453 0.386 no no 0.513 50 3.289 

High-flow 

All 0.415 0.390 0.025 5.153 5.179 194 0.012 yes no 0.212 50 1.875 NO3

-

< 0.1 0.446 0.456 -0.010 0.526 0.535 71 0.341 no no 0 0.094 0.007 

0.1 < NO3

-

≤ 1.0 0.125 0.106 0.019 0.029 0.020 60 0.229 no no 0.372 0.984 0.795 

NO3

- > 1.0 0.657 0.588 0.069 15.278 15.375 63 0.004 yes yes 3.536 50 6.27 

Low-flow 

All 0.283 0.290 -0.007 0.319 0.321 259 0.540 no no 0.674 48 3.96 NO3

- < 0.1 0.466 0.494 -0.027 0.708 0.698 82 0.018 yes no 0.02 0.084 0.04 

0.1 < NO3

-

≤ 1.0 0.107 0.100 0.007 0.096 0.099 60 0.303 no no 0.467 0.988 0.628 

NO3

- > 1.0 0.245 0.244 0.000 0.124 0.127 117 0.993 no no 4.222 48 8.6 

Filtered 

surface water 

All 0.41 0.435 -0.025 0.191 0.226 543 <0.001 yes yes 0.182 16.88 0.883 

High-flow 

All 0.385 0.393 -0.008 0.078 0.088 295 0.21 no no 0.358 16.88 0.922 NO3

-

 < 0.1 0.365 0.34 0.025 0.082 0.088 95 0.041 yes no 0.016 0.096 0.053 

0.1 < NO3

-

≤ 1.0 0.403 0.401 0.002 0.073 0.081 136 0.836 no no 0.329 1.547 0.541 

NO3

- > 1.0 0.388 0.456 -0.068 0.084 0.096 64 0.002 yes yes 2.111 16.88 3.199 

Low-flow 

All 0.44 0.484 -0.044 0.325 0.387 248 <0.001 yes yes 0.084 9.76 0.475 NO3

- < 0.1 0.436 0.462 -0.026 0.226 0.232 131 <0.001 yes yes 0.009 0.098 0.027 

0.1 < NO3

-

≤ 1.0 0.359 0.374 -0.015 0.14 0.128 75 0.342 no no 0.236 0.967 0.468 

NO3

- > 1.0 0.599 0.748 -0.149 0.95 1.279 42 <0.001 yes yes 2.238 9.757 3.819 
 28 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

Table 15. Results of paired t-tests for nitrate-corrected alkaline persulfate total nitrogen and Kjeldahl total nitrogen concentrations determined in acidified whole-water samples. Distributions of nitrate concentrations for each subgroup are shown in the rightmost three columns[mg-N/L, milligrams nitrogen per liter; TNAlkP, alkaline persulfate digestion total nitrogen; KTN, Kjeldahl digestion total nitrogen; n, number of samples; max, maximum; 75th, 75th percentile; <, less than; <, less than or equal to; >, greater than]

 1pcalc is the probability that population means of nitrate-corrected TNAlkP and KTN concentrations are the same—that is, difference between the population means is statistically 

equivalent to zero—on the basis of calculated paired t-tests. Difference between population means is significant at the 95-percent confidence level (p0.05) when pcalc is less than 0.05 and at the 99-percent confidence level (p0.01) when pcalc is less than 0.01.

Mean 

(mg-N/L) Variance (mg-N/L) Significance1 Nitrate (mg-N/L) Unfiltered ground water TNAlkP KTN Difference (mg-N/L) TNAlkP KTN n

pcalc p0.05 p0.01 Median Max 75th 

All 0.514 0.577 -0.063 0.319 0.362 111 <0.001 yes yes 0.022 16.04 0.859 

High-flow 

All 0.509 0.53 -0.021 0.178 0.179 48 0.135 no no 0.498 16.04 0.868 NO3

-

 < 0.1 0.242 0.277 -0.035 0.162 0.181 23 0.001 yes yes 0.010 0.052 0.017 

0.1 < NO3

-

≤ 1.0 0.821 0.795 0.026 0.034 0.032 21 0.096 no no 0.864 0.904 0.872 

NO3

- > 1.0 0.414 0.593 -0.179 0.135 0.285 4 0.185 no no 4.164 16.04 8.613 

Low-flow 

All 0.518 0.613 -0.095 0.431 0.503 63 <0.001 yes yes 0.017 6.962 0.052 NO3

-

 < 0.1 0.596 0.679 -0.083 0.51 0.613 48 <0.001 yes yes 0.015 0.062 0.022 

0.1 < NO3

-

≤ 1.0 0.429 0.575 -0.146 0.114 0.097 7 0.127 no no 0.344 0.885 0.866 

NO3

- > 1.0 0.092 0.211 -0.119 0.062 0.062 8 <0.001 yes yes 1.797 6.962 5.376 

Unfiltered 

surface water 

All 0.728 0.820 -0.092 0.911 1.007 959 <0.001 yes yes 0.215 61.65 0.874 

High-flow 

All 0.677 0.744 -0.067 0.608 0.694 468 <0.001 yes yes 0.345 11.74 0.993 NO3

-

 < 0.1 0.654 0.655 -0.001 0.560 0.517 116 <0.001 no no 0.020 0.095 0.054 

0.1 < NO3

-

≤ 1.0 0.573 0.625 -0.052 0.331 0.380 237 <0.001 yes yes 0.349 0.999 0.585 

NO3

-

> 1.0 0.914 1.080 -0.166 1.160 1.385 115 <0.001 yes yes 2.119 11.74 3.125 

Low-flow 

All 0.778 0.892 -0.114 1.197 1.297 491 <0.001 yes yes 0.134 61.65 0.807 NO3

- < 0.1 0.898 0.974 -0.076 1.303 1.374 217 <0.001 yes yes 0.034 0.099 0.057 

0.1 < NO3

-

≤ 1.0 0.588 0.689 -0.101 0.299 0.395 177 <0.001 yes yes 0.330 0.974 0.557 

NO3

-

 > 1.0 0.855 1.076 -0.221 2.524 2.681 97 <0.001 yes yes 2.380 61.65 4.648 
Summary and Conclusions 29

in this report were obtained from samples collected 

during nominally high-flow (April–June 2002) 

conditions, and the other half were collected during 

nominally low-flow (August–September 2002) 

conditions. Numbers of filtered and acidified wholewater samples were about equal. This report provides 

details of alkaline persulfate digest preparation as well 

as complete operational information, including 

interferences and analytical figures of merit for the 

automated colorimetric methods developed to 

determine nitrate and orthophosphate in alkaline 

persulfate digests. Primary conclusions of this report 

follow:

1. Hazards to analysts and toxic wastes are 

substantially less for alkaline persulfate digestion 

methods than for Kjeldahl digestion methods.

2. Alkaline persulfate digestion methods described in 

this report can be applied successfully to acidified 

samples (USGS FCA and WCA bottle types) 

provided that samples are acidified at collection 

sites using supplies and protocols specified in the 

USGS field manual (Wilde and others, 1998).

3. Alkaline persulfate digestion methods described in 

this report are amenable to automation and should 

prove substantially less labor intensive than the 

existing Kjeldahl digestion methods. For 

example, filtered-water sample digests can be 

prepared robotically, and the manual 

.005 0.01 0.1 1 5

.005

0.01

0.1

1

5

Line of equal relation

n = 2093

KJELDAHL PHOSPHORUS, IN

MILLIGRAMS PHOSPHORUS PER LITER

ALKALINE PERSULFATE PHOSPHORUS, IN

MILLIGRAMS PHOSPHORUS PER LITER

Figure 10. Logarithmic scatter plot of PAlkP concentrations (x-axis) and KP concentrations (y-axis) around 

the line of equal relation for 2,093 data pairs combined from large-scale validation experiments. The linear 

regression equation for these data is KP = (0.994 ± 0.002) PAlkP + 0.0003 ± 0.0005, with a correlation 

coefficient (r2) of 0.994.
 30 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

Table 16. Results of paired t-tests for dissolved and total phosphorus concentrations determined in filtered and acidified whole-water samples by alkaline persulfate digestion (PAlkP) and Kjeldahl digestion (KP) methods[mg-P/L, milligrams phosphorus per liter; n, number of samples; FCC, filtered, chilled (bottle type for samples); WCA, whole water, chilled, acidified (bottle type for samples); MC 6, 

ground water medium code; MC 9, surface water medium code; HF, high flow; LF, low flow; <, less than]

1pcalc is the probability that population means of PAlkP and KP concentrations are the same—that is, difference between the population means is statistically equivalent to zero—on 

the basis of calculated paired t-tests. Difference between population means is significant at the 95-percent confidence level (p0.05) when pcalc is less than 0.05 and at the 99-percent 

confidence level (p0.01) when pcalc is less than 0.01.

 Mean (mg-P/L) Variance (mg-P/L) Significance1 Water 

type 

PAlkP KP 

Difference 

(mg-P/L) 

PAlkP KP 

n pcalc p0.05 p0.01

All 0.134 0.133 0.001 0.061 0.060 2,093 0.242 no no All FCC 0.100 0.103 -0.003 0.063 0.062 1,115 <0.0001 yes yes All WCA 0.171 0.167 0.004 0.055 0.056 978 <0.0001 yes yes All MC 6 0.084 0.087 -0.003 0.045 0.045 645 <0.0001 yes yes All MC 9 0.155 0.154 0.002 0.066 0.066 1,448 <0.0005 yes yes 

High-flow 

All 0.123 0.123 0.000 0.045 0.046 990 0.640 no no FCC, MC 6 0.068 0.069 -0.001 0.026 0.028 204 0.557 no no FCC, MC 9 0.126 0.131 -0.006 0.059 0.061 320 <0.0001 yes yes WCA, MC 6 0.296 0.297 -0.001 0.124 0.125 49 0.787 no no WCA, MC 9 0.127 0.123 0.004 0.028 0.029 417 <0.0001 yes yes 

Low-flow 

All 0.143 0.142 0.001 0.075 0.073 1,103 0.043 yes no FCC, MC 6 0.046 0.050 -0.004 0.023 0.022 337 <0.0005 yes yes FCC, MC 9 0.166 0.166 0.000 0.141 0.135 254 0.833 no no WCA, MC 6 0.190 0.193 -0.003 0.116 0.119 55 0.140 no no WCA, MC 9 0.196 0.190 0.006 0.062 0.063 457 <0.0001 yes yes 
Summary and Conclusions 31

post-digestion, pH adjustment step typical in 

previously reported alkaline persulfate digestion 

procedures (Ameel and others, 1993; D’Elia and 

others, 1997) has been eliminated.

4. Method detection limits (MDLs) for the alkaline 

persulfate digestion dissolved and total nitrogen 

(0.015 mg-N/L) and phosphorus (0.007 mg-P/L) 

are substantially less than those of USGS methods 

I-2515/4515/91 for dissolved and total Kjeldahl 

nitrogen (0.05 mg-N/L) and USGS methods 

I-2610/4610/91 for dissolved and total Kjeldahl 

phosphorus (0.02 mg-P/L) methods. The lower 

nitrogen and phosphorus MDLs of alkaline 

persulfate digestion methods described in this 

report improve the precision of nutrient-mass 

balance estimates.

5. On the basis of two-population, paired t-test 

statistics, the means of all nitrate-corrected 

alkaline persulfate digestion nitrogen (NAlkP) and 

Kjeldahl digestion nitrogen (KN) concentrations 

(2,066 paired results) were significantly different 

from zero at the p = 0.05 level. Statistical and 

graphical analyses of experimental data indicate 

that concentration differences between nitratecorrected NAlkP and KN result from nitrate 

interference in the Kjeldahl digestion method 

rather than incomplete recovery of nitrogen by the 

alkaline persulfate digestion method. Alkaline 

persulfate digestion, therefore, provides more 

accurate estimates of total nitrogen 

concentrations in samples that contain nitrate 

concentrations greater than about 0.1 mg 

NO3

-

-N/L. For some subsets of these data, the 

means were not different from zero at the p = 0.05 

level, typically in ground-water samples or in 

surface-water samples with nitrate concentrations 

less than 0.1 mg-N/L.

6. On the basis of two-population, paired t-test 

statistics for 2,093 paired results, the means of all 

Kjeldahl digestion phosphorus concentrations 

determined by USGS method I-2610/4610-91 

(similar to USEPA method 365.4) and those 

determined by the alkaline persulfate digestion 

method reported here were not significantly 

different from zero at the p = 0.05 level. For some 

subsets of these data, the means were different 

from zero at the p = 0.05 level, but in such cases 

differences were less than the method detection 

limit (0.007 mg-P/L) for the alkaline persulfate 

digestion method and were not analytically 

significant. Changing from Kjeldahl digestion to 

alkaline persulfate digestion, therefore, does not 

affect comparisons with historical dissolved and 

total phosphorus concentrations.

7. Data and analysis provided in this report establish 

guidelines necessary to interpret total and 

dissolved nitrogen and phosphorus concentrations 

that result from alkaline persulfate digestion 

methods in relation to those that result from 

Kjeldahl digestion methods. Specifically—

a. Systematic differences between DNAlkP /KDN 

and TNAlkP /KTN concentrations are 

expected for samples with dissolved nitrate 

concentrations greater than or equal to 

0.1 mg-N/L. 

b. Concentration differences between NAlkP and 

historical KN data are likely to increase in 

proportion to dissolved nitrate 

concentrations in samples. Whether 

concentration differences are positive or 

negative depend on water type in ways that 

were not possible to describe fully.

(1) Negative differences between KN and

 NAlkP were found most often for

 surface-water samples and unfiltered

 ground-water samples.

(2) Differences between KN and NAlkP in

 filtered ground water are as likely to

 be negative as positive.

c. Samples with organic carbon (OC) 

concentrations greater than about 150 mg/L 

are not amenable to NAlkP determinations 

unless OC concentrations are diluted below 

this threshold prior to digestion.

d. As nitrate concentrations increase, NAlkP 

digestion provides better estimates of total 

and dissolved nitrogen than KN digestion, 

which suffers from positive and negative 

interference by nitrate. On the other hand, 

estimating organic nitrogen concentrations 

as the small difference between two large 

numbers when dissolved nitrate, and 

therefore NAlkP , concentrations are large 

also can be problematic.

e. Systematic concentration differences between 

PAlkP and historical KP data are not 

expected.

8. One major conclusion of this report—that alkaline 

persulfate digestion is a more sensitive, accurate, 

and environmentally responsible alternative to 
32 EVALUATION OF ALKALINE PERSULFATE DIGESTION AS AN ALTERNATIVE TO KJELDAHL DIGESTION

FOR DETERMINATION OF TOTAL AND DISSOLVED NITROGEN AND PHOSPHORUS IN WATER

Kjeldahl digestion for routine, simultaneous 

determination of nitrogen and phosphorus in 

surface and ground water—is consistent with 

previously published studies that are cited 

throughout this report. In comparison to these 

earlier studies, however, conclusions of this report 

are based on a much larger and geographically 

diverse sample population collected during highflow and low-flow conditions. Furthermore, 

samples were collected, preserved, stored, and 

analyzed by rigorously controlled protocols 

established and documented by the USGS. In 

these respects, this report describes the most 

comprehensive study to date supporting 

applicability of the alkaline persulfate digestion 

method as a superior alternative to the time 

honored, but operationally flawed, Kjeldahl 

digestion method.

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