5530 PHENOLS*

5530 A. Introduction

Phenols, defined as hydroxy derivatives of benzene and its by acidification. Some highly contaminated wastewaters may
condensed nuclei, may occur in domestic and industrial waste- require specialized techniques for eliminating interferences and
waters, natural waters, and potable water supplies. Chlorination for quantitative recovery of phenolic compounds.
of such waters may produce odorous and objectionable-tasting Eliminate major interferences as follows (see Section 5530B
chlorophenols. Phenol removal processes in water treatment for reagents):
include superchlorination, chlorine dioxide or chloramine treat- Oxidizing agents, such as chlorine and those detected by the
ment, ozonation, and activated carbon adsorption. liberation of iodine on acidification in the presence of potassium
iodide (KI)—Remove immediately after sampling by adding
1. Selection of Method excess ferrous sulfate (FeSO4). If oxidizing agents are not re-
moved, the phenolic compounds will be oxidized partially.
The analytical procedures offered here use the 4-aminoan- Sulfur compounds—Remove by acidifying to pH 4.0 with
tipyrine colorimetric method that determines phenol, ortho- and H3PO4 and aerating briefly by stirring. This eliminates the in-
meta-substituted phenols, and, under proper pH conditions, those terference of hydrogen sulfide (H2S) and sulfur dioxide (SO2).
para-substituted phenols in which the substitution is a carboxyl, Oils and tars—Make an alkaline extraction by adjusting to pH 12
halogen, methoxyl, or sulfonic acid group. The 4-aminoan- to 12.5 with NaOH pellets. Extract oil and tar from aqueous solution
tipyrine method does not determine those para-substituted phe- with 50 mL chloroform (CHCl3). Discard oil- or tar-containing
nols where the substitution is an alkyl, aryl, nitro, benzoyl, layer. Remove excess CHCl3 in aqueous layer by warming on a
nitroso, or aldehyde group. A typical example of these latter water bath before proceeding with the distillation step.
groups is paracresol, which may be present in certain industrial
wastewaters and in polluted surface waters.
3. Sampling
The 4-aminoantipyrine method is given in two forms: Method
C, for extreme sensitivity, is adaptable for use in water samples
Sample in accordance with the instructions of Section 1060.
containing less than 1 mg phenol/L. It concentrates the color in
a nonaqueous solution. Method D retains the color in the aque-
ous solution. Because the relative amounts of various phenolic 4. Preservation and Storage of Samples
compounds in a given sample are unpredictable, it is not possible
to provide a universal standard containing a mixture of phenols. Phenols in concentrations usually encountered in wastewaters are
For this reason, phenol (C6H5OH) itself has been selected as a subject to biological and chemical oxidation. Preserve and store
standard for colorimetric procedures and any color produced by samples at 4°C or lower unless analyzed within 4 h after collection.
the reaction of other phenolic compounds is reported as phenol. Acidify with 2 mL conc H2SO4/L.
Because substitution generally reduces response, this value rep- Analyze preserved and stored samples within 28 d after col-
resents the minimum concentration of phenolic compounds. A lection.
gas-liquid chromatographic procedure is included in Section
6420B and may be applied to samples or concentrates to quantify 5. Bibliography
individual phenolic compounds.
ETTINGER, M.B., S. SCHOTT & C.C. RUCHHOFT. 1943. Preservation of
2. Interferences phenol content in polluted river water samples previous to analysis.
J. Amer. Water Works Assoc. 35:299.
Interferences such as phenol-decomposing bacteria, oxidizing CARTER, M.J. & M.T. HUSTON. 1978. Preservation of phenolic com-
and reducing substances, and alkaline pH values are dealt with pounds in wastewaters. Environ. Sci. Technol. 12:309.
NEUFELD, R.D. & S.B. POLADINO. 1985. Comparison of 4-aminoan-
tipyrine and gas-liquid chromatography techniques for analysis of
* Approved by Standard Methods Committee, 2005. phenolic compounds. J. Water Pollut. Control Fed. 57:1040.

5530 B. Cleanup Procedure

1. Principle 2. Apparatus
Phenols are distilled from nonvolatile impurities. Because the
a. Distillation apparatus, all-glass, consisting of a 1-L boro-
volatilization of phenols is gradual, the distillate volume must
silicate glass distilling apparatus with Graham condenser.*
ultimately equal that of the original sample. b. pH meter.

* Corning No. 3360 or equivalent.

1
 PHENOLS (5530)/Chloroform Extraction Method

3. Reagents b. Distill 450 mL, stop distillation and, when boiling ceases,
add 50 mL warm water to distilling flask. Continue distillation
Prepare all reagents with distilled water free of phenols and until a total of 500 mL has been collected.
chlorine. c. One distillation should purify the sample adequately. Oc-
a. Phosphoric acid solution, H3PO4, 1 ⫹ 9: Dilute 10 mL 85% casionally, however, the distillate is turbid. If so, acidify with
H3PO4 to 100 mL with water. H3PO4 solution and distill as described in ¶ 4b. If second
b. Methyl orange indicator solution. distillate is still turbid, use extraction process described in ¶ 4d
c. Special reagents for turbid distillates: before distilling sample.
1) Sulfuric acid, H2SO4, 1N. d. Treatment when second distillate is turbid: Extract a
2) Sodium chloride, NaCl. 500-mL portion of original sample as follows: Add 4 drops
3) Chloroform, CHCl3, or methylene chloride, CH2Cl2. methyl orange indicator and make acidic to methyl orange with
4) Sodium hydroxide, NaOH, 2.5N: Dilute 41.7 mL 6N NaOH 1N H2SO4. Transfer to a separatory funnel and add 150 g NaCl.
to 100 mL or dissolve 10 g NaOH pellets in 100 mL water. Shake with five successive portions of CHCl3, using 40 mL in
the first portion and 25 mL in each successive portion. Transfer
CHCl3 layer to a second separatory funnel and shake with three
4. Procedure successive portions of 2.5N NaOH solution, using 4.0 mL in the
first portion and 3.0 mL in each of the next two portions.
a. Measure 500 mL sample into a beaker, adjust pH to ap- Combine alkaline extracts, heat on a water bath until CHCl3 has
proximately 4.0 with H3PO4 solution using methyl orange indi- been removed, cool, and dilute to 500 mL with distilled water.
cator or a pH meter, and transfer to distillation apparatus. Use a Proceed with distillation as described in ¶s 4a and b.
500-mL graduated cylinder as a receiver. Omit adding H3PO4 NOTE: CH2Cl2 may be used instead of CHCl3, especially if an
and adjust pH to 4.0 with 2.5N NaOH if sample was preserved emulsion forms when the CHCl3 solution is extracted with
as described in 5530A.4. NaOH.

5530 C. Chloroform Extraction Method

1. General Discussion d. pH meter.

e. Separatory funnels, 1000-mL, Squibb form, with ground-
a. Principle: Steam-distillable phenols react with 4-aminoan- glass stoppers and TFE stopcocks. At least eight are required.
tipyrine at pH 7.9 ⫾ 0.1 in the presence of potassium ferricya-
nide to form a colored antipyrine dye. This dye is extracted from 3. Reagents
aqueous solution with CHCl3 and the absorbance is measured at
460 nm. This method covers the phenol concentration range Prepare all reagents with distilled water free of phenols and
from 1.0 ␮g/L to over 250 ␮g/L with a sensitivity of 1 ␮g/L. chlorine.
b. Interference: All interferences are eliminated or reduced to a. Stock phenol solution: Dissolve 100 mg phenol in freshly
a minimum if the sample is preserved, stored, and distilled in boiled and cooled distilled water and dilute to 100 mL. CAU-
accordance with the foregoing instructions. TION—Toxic; handle with extreme care. Ordinarily this direct
c. Minimum detectable quantity: The minimum detectable weighing yields a standard solution; if extreme accuracy is
quantity for clean samples containing no interferences is 0.5 ␮g required, standardize as follows:
phenol when a 25-mL CHCl3 extraction with a 5-cm cell or a 1) To 100 mL water in a 500-mL glass-stoppered conical flask,
50-mL CHCl3 extraction with a 10-cm cell is used in the pho- add 50.0 mL stock phenol solution and 10.0 mL bromate-
tometric measurement. This quantity is equivalent to 1 ␮g phe- bromide solution. Immediately add 5 mL conc HCl and swirl
nol/L in 500 mL distillate. gently. If brown color of free bromine does not persist, add
10.0-mL portions of bromate-bromide solution until it does.
2. Apparatus Keep flask stoppered and let stand for 10 min; then add approx-
imately 1 g KI. Usually four 10-mL portions of bromate-bromide
a. Photometric equipment: A spectrophotometer for use at 460 solution are required if the stock phenol solution contains 1000
nm equipped with absorption cells providing light paths of 1 to mg phenol/L.
10 cm, depending on the absorbances of the colored solutions 2) Prepare a blank in exactly the same manner, using distilled
and the individual characteristics of the photometer. water and 10.0 mL bromate-bromide solution. Titrate blank and
b. Filter funnels: Buchner type with fritted disk.* sample with 0.025M sodium thiosulfate, using starch solution
c. Filter paper: Alternatively use an appropriate 11-cm filter indicator.
paper for filtering CHCl3 extracts instead of the Buchner-type 3) Calculate the concentration of phenol solution as follows:
funnels and anhydrous Na2SO4.
mg phenol/L ⫽ 7.842 [(A ⫻ B) ⫺ C]

* 15-mL Corning No. 36060 or equivalent. where:

2
 PHENOLS (5530)/Chloroform Extraction Method

A ⫽ mL thiosulfate for blank, tion. Construct a separate calibration curve for each photometer
B ⫽ mL bromate-bromide solution used for sample divided by and check each curve periodically to insure reproducibility.
10, and b. For infrequent analyses prepare only one standard phenol
C ⫽ mL thiosulfate used for sample. solution. Prepare 500 mL standard phenol solution of a strength
approximately equal to the phenolic content of that portion of
b. Intermediate phenol solution: Dilute 1.00 mL stock phenol original sample used for final analysis. Also prepare a 500-mL
solution in freshly boiled and cooled distilled water to 100 mL; distilled water blank.
1 mL ⫽ 10.0 ␮g phenol. Prepare daily. Continue as described in ¶ a, above, but measure absorbances
c. Standard phenol solution: Dilute 50.0 mL intermediate of sample and standard phenol solution against the blank at 460
phenol solution to 500 mL with freshly boiled and cooled dis- nm.
tilled water; 1 mL ⫽ 1.0 ␮g phenol. Prepare within 2 h of use.
d. Bromate-bromide solution: Dissolve 2.784 g anhydrous 5. Calculation
KBrO3 in water, add 10 g KBr crystals, dissolve, and dilute to
1000 mL. a. For Procedure a:
e. Hydrochloric acid, HCl, conc.
f. Standard sodium thiosulfate titrant, 0.025M: See Section
4500-O.C.2e. A
␮g phenol/L ⫽ ⫻ 1000
g. Starch solution: See Section 4500-O.C.2d. B
h. Ammonium hydroxide, NH4OH, 0.5N: Dilute 35 mL fresh,
conc NH4OH to 1 L with water. where:
i. Phosphate buffer solution: Dissolve 104.5 g K2HPO4 and A ⫽ ␮g phenol in sample, from calibration curve, and
72.3 g KH2PO4 in water and dilute to 1 L. The pH should be 6.8. B ⫽ mL original sample.
j. 4-Aminoantipyrine solution: Dissolve 2.0 g 4-aminoan-
tipyrine in water and dilute to 100 mL. Prepare daily. b. For Procedure b, calculate the phenol content of the orig-
k. Potassium ferricyanide solution: Dissolve 8.0 g K3Fe(CN)6 inal sample:
in water and dilute to 100 mL. Filter if necessary. Store in a
brown glass bottle. Prepare fresh weekly. C ⫻ D ⫻ 1000
l. Chloroform, CHCl3. ␮g phenol/L ⫽
E⫻B
m. Sodium sulfate, anhydrous Na2SO4, granular.
n. Potassium iodide, KI, crystals.
where:
4. Procedure C ⫽ ␮g standard phenol solution,
D ⫽ absorbance reading of sample,
E ⫽ absorbance of standard phenol solution, and
Ordinarily, use Procedure a; however, Procedure b may be
B ⫽ mL original sample.
used for infrequent analyses.
a. Place 500 mL distillate, or a suitable portion containing not
more than 50 ␮g phenol, diluted to 500 mL, in a 1-L beaker. 6. Precision and Bias
Prepare a 500-mL distilled water blank and a series of 500-mL
phenol standards containing 5, 10, 20, 30, 40, and 50 ␮g phenol. Because the “phenol” value is based on C6H5OH, this method
Treat sample, blank, and standards as follows: Add 12.0 mL yields only an approximation and represents the minimum
0.5N NH4OH and immediately adjust pH to 7.9 ⫾ 0.1 with amount of phenols present. This is true because the phenolic
phosphate buffer. Under some circumstances, a higher pH may reactivity to 4-aminoantipyrine varies with the types of phenols
be required.† About 10 mL phosphate buffer are required. Trans- present.
fer to a 1-L separatory funnel, add 3.0 mL aminoantipyrine In a study of 40 refinery wastewaters analyzed in duplicate at
solution, mix well, add 3.0 mL K3Fe(CN)6 solution, mix well, concentrations from 0.02 to 6.4 mg/L the average relative stan-
and let color develop for 15 min. The solution should be clear dard deviation was ⫾ 12%. Data are not available for precision
and light yellow. at lower concentrations.
Extract immediately with CHCl3, using 25 mL for 1- to 5-cm
cells and 50 mL for a 10-cm cell. Shake separatory funnel at least 7. Bibliography
10 times, let CHCl3 settle, shake again 10 times, and let CHCl3
settle again. Filter each CHCl3 extract through filter paper or SCOTT, R.D. 1931. Application of a bromine method in the determination
fritted glass funnels containing a 5-g layer of anhydrous Na2SO4. of phenols and cresols. Ind. Eng. Chem., Anal. Ed. 3:67.
Collect dried extracts in clean cells for absorbance measure- EMERSON, E., H.H. BEACHAM & L.C. BEEGLE. 1943. The condensation of
ments; do not add more CHCl3 or wash filter papers or funnels aminoantipyrine. II. A new color test for phenolic compounds. J.
with CHCl3. Org. Chem. 8:417.
ETTINGER, M.B. & R.C. KRONER. 1949. The determination of phenolic
Read absorbance of sample and standards against the blank at
materials in industrial wastes. Proc. 5th Ind. Waste Conf., Purdue
460 nm. Plot absorbance against micrograms phenol concentra- Univ., p. 345.
ETTINGER, M.B., C.C. RUCHHOFT & R.J. LISHKA. 1951. Sensitive 4-ami-
noantipyrine method for phenolic compounds. Anal. Chem. 23:
† For NPDES permit analyses, pH 10 ⫾ 0.1 is required. 1783.

3
 PHENOLS (5530)/Direct Photometric Method

DANNIS, M. 1951. Determination of phenols by the aminoantipyrine FAUST, S.D. & O.M. ALY. 1962. The determination of 2,4-dichlorophe-
method. Sewage Ind. Wastes 23:1516. nol in water. J. Amer. Water Works Assoc. 54:235.
MOHLER, E.F., JR. & L.N. JACOB. 1957. Determination of phenolic-type FAUST, S.D. & E.W. MIKULEWICZ. 1967. Factors influencing the conden-
compounds in water and industrial waste waters: Comparison of sation of 4-aminoantipyrine with derivatives of hydroxybenzene. II.
analytical methods. Anal. Chem. 29:1369. Influence of hydronium ion concentration on absorptivity. Water
BURTSCHELL, R.H., A.A. ROSEN, F.M. MIDDLETON & M.B. ETTINGER. Res. 1:509.
1959. Chlorine derivatives of phenol causing taste and odor. FAUST, S.D. & P.W. ANDERSON. 1968. Factors influencing the conden-
J. Amer. Water Works Assoc. 51:205. sation of 4-aminoantipyrine with derivations of hydroxy benzene.
GORDON, G.E. 1960. Colorimetric determination of phenolic materials in III. A study of phenol content in surface waters. Water Res. 2:515.
refinery waste waters. Anal. Chem. 32:1325. SMITH, L.S. 1976. Evaluation of Instrument for the (Ultraviolet) Deter-
OCHYNSKI, F.W. 1960. The absorptiometric determination of phenol. mination of Phenol in Water. EPA-600/4-76-048, U.S. Environ-
Analyst 85:278. mental Protection Agency, Cincinnati, Ohio.

5530 D. Direct Photometric Method

1. General Discussion NH4OH solution and immediately adjust to pH 7.9 ⫾ 0.1 with
phosphate buffer. Add 1.0 mL 4-aminoantipyrine solution, mix
a. Principle: Steam-distillable phenolic compounds react with well, add 1.0 mL K3Fe(CN)6 solution, and mix well.
4-aminoantipyrine at pH 7.9 ⫾ 0.1 in the presence of potassium After 15 min, transfer to cells and read absorbance of sample
ferricyanide to form a colored antipyrine dye. This dye is kept in and standards against the blank at 500 nm.
aqueous solution and the absorbance is measured at 500 nm.
b. Interference: Interferences are eliminated or reduced to a 5. Calculation
minimum by using the distillate from the preliminary distillation
procedure. a. Use of calibration curve: Estimate sample phenol content
c. Minimum detectable quantity: This method has less sensi- from photometric readings by using a calibration curve con-
tivity than Method C. The minimum detectable quantity is 10 ␮g structed as directed in Section 5530C.4a.
phenol when a 5-cm cell and 100 mL distillate are used.
A
mg phenol/L ⫽ ⫻ 1000
2. Apparatus B

where:
a. Photometric equipment: Spectrophotometer equipped with
A ⫽ mg phenol in sample, from calibration curve, and
absorption cells providing light paths of 1 to 5 cm for use at 500
B ⫽ mL original sample.
nm.
b. pH meter. b. Use of single phenol standard:

3. Reagents C ⫻ D ⫻ 1000
mg phenol/L ⫽
E⫻B
See Section 5530C.3.
where:
C ⫽ mg standard phenol solution,
4. Procedure
D ⫽ absorbance of sample, and
E ⫽ absorbance of standard phenol solution.
Place 100 mL distillate, or a portion containing not more than
0.5 mg phenol diluted to 100 mL, in a 250-mL beaker. Prepare
a 100-mL distilled water blank and a series of 100-mL phenol 6. Precision and Bias
standards containing 0.1, 0.2, 0.3, 0.4, and 0.5 mg phenol. Treat
sample, blank, and standards as follows: Add 2.5 mL 0.5N Precision and bias data are not available.