foO 0 00 0 4 9

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R E S E A R C H T R I A N G L E I N S T I T U T E

ISOLATION AND IDENTIFICATION OF CONTAMINANT(S) IN GROUND WATER

RTI No.: 321T-3678

FINAL REPORT

Analytical and Chemical SciencesResearch Triangle Institute

P. 0. Box 12194Research Triangle Park, NC 27709

Prepared by: Approved by:

P. A. HyIdBurg, Ph.*. E. D. PellizzaFi, Ph.D.VIce-President, Analyticaland Chemical Sciences

December 1986

PREPARED FOR

Environmental and Safety Designs, Inc.Post Office Box 341315

Memphis, Tennessee 38184

P O S T O F F I C E B O X 1 2 1 9 4 R E S E A R C H T R I A N G L E P A R K , N O R T H C A R O L I N A 2 7 7 0 9

EPA Region 5 Records Ctr.

209141

TABLE OF CONTENTS

Page

ABSTRACT • 11

1.0 INTRODUCTION 1

2.0 TECHNICAL DISCUSSION 2

3.0 CONCLUSIONS 14

4.0 RECOMMENDATIONS 40

5.0 REFERENCES 40

APPENDIX A - PROTOCOLS FOR ANALYSISPROTOCOL A: SORBENT ACCUMULATOR COLUMN (SC) EXTRACTION 43

PROTOCOl B: C: EANUP/FRACTIONATION 51

ABSTRACT

The purpose of this project was to Isolate and Identify compound(s)

responsible for positive phenols test (4-aminoantipyrine method) in

groundwater surrounding area used as a waste site for wood tar wastes

between 1910 and 1945. Following the failure of standard analytical

techniques such as liquid-liquid extraction and GC/MS to isolate the target

analyte(s), a sorbent accumulator column (XAD-4) technique was tried and

modified for the purpose of this project. Extracts generated by this

method were analyzed by GC/MS, and a series of alkyl phenols were

identified at sub-ppb levels.

ii

1.0 INTRODUCTION

This final report summarizes work performed by Research Triangle Insti-

tute (RTI) for Environmental and Safety Designs, Inc. (EnSafe), Memphis,

Tennessee under Contract DTD.7-14-86.

EnSafe, under contract to the various interested parties, has been

involved in the Investigation of and possible remedial alternative for the

Antrim Iron Works Site (a.k.a. Tar Lake) 1n Antrim County, Michigan. The

Antrim Iron Works Site was subjected to releases of wood tar wastes between

1910 and 1945. The site has been Idle since 1945. Detailed information

regarding the site, the wastes discharged, hydrogeologlcal and topographi-

cal aspects of the area, and monitoring results from the site was provided

by EnSafe 1n the form of a Work Plan, which they prepared for the owners of

the site.

EnSafe had observed that a constantly-enlarging "plume" of groundwater

contaminated with an unknown chemical was present 1n the area of Tar Lake.

Samples from the plume yielded positive results for the 4-aminoantipyrine

test used for identification of phenols (1). This test yielded results in

the low to middle ppb level. Groundwater samples from this area also

exhibited high (ppm) TOC levels and low dissolved oxygen concentrations.

Attempts were made to Identify the contamlnant(s) using standard analytical

techniques (I.e., liquid-liquid extraction followed by GC/MS analysis).

Inconsistent and Inconclusive results were obtained, and no compounds were

identified that wo'.:ld account for the results of the 4-aminoantipyrine

test. The objective of the work described herein was to isolate and iden-

tify the compound(s) responsible for this positive test.

2.0 TECHNICAL DISCUSSION

Prior to any sample collection or analysis, a computer-assisted litera-

ture search was performed to determine all analytes that could produce a

positive response to the 4-aminoant1pyr1ne test for total phenols. No

information was found other than that contained in Method 510, Phenols in

Standard Methods (1), which describes In detail the 4-am1noant1pyrine

colorimetric test for phenols. In Method 510, it states that (a) oxidizing

agents can decompose phenols 1f analysis is delayed more than four hours

after sample collection, and (b) sulfur compounds can produce false posi-

tive readings. Method 510 recommends the use of ferrous sulfate to prevent

oxidation and the addition of phosphoric add followed by brief aeration to

eliminate interferences caused by sulfur-containing compounds.

The first sampling for the purposes of this contract was performed on

July 30, 1986. Water was collected from well number 7 of the Tar Lake

site. After first flushing the well housing, several liters of water were

collected using each of three preservation methods: (1) no preservative

added, (2) 2 mL of phosphoric add (50%) added to each liter, and (3) 2 mL

of phosphoric add plus 5 g of ferrous sulfate. All samples were

refrigerated from the time of collection until the time of final analysis

and were shipped, stored, and analyzed In effective darkness (yellow lights

only) to prevent possible photodegradation. An aliquot of water with no

preservatives added was analyzed at the time of collection for total

phenols by the 4-am1noant1pyrine method. This determination yielded a

results of 14 ppb total phenols.

Immediately on receipt of the water samples at RTI (August 1, 1986),

the 4-aminoantipyrine test was repeated on aliquots preserved by each of

the three methods listed above. All aliquots yielded results of approxima-

tely 5 ppb, Indicating, first, that the analyte(s) responsible for the

positive 4-aminoant1pyr1ne test at the sampling site was still present

after shipping (although at a lower level), and, second, that the various

methods of preservation had no effect.

Total Organic Carbon (TOC) was determined on an aliquot to which no

preservatives had been added and found to be approximately 7 ppm.

Dissolved oxygen was determined to be 2.5 ppm in an aliquot with no preser-

vative added and 5.5 ppm In an aliquot with both phosphoric add and

ferrous sulfate added. This higher dissolved oxygen value in the sample

taken with preservatives was unexpected and may derive from the slightly

increased manipulation to which that sample was subjected during the addi-

tion of the preservatives.

An ultraviolet (UV) spectrum was taken on an aliquot of water sample

with no preservatives added. A Perkln Elmer Lambda 3 Spectrophotometer was

used at maximum sensitivity. No absorbance was noted In the UV range from

750 to 190 nm, as determined versus a delonlzed water blank.

One liter aliquots of sample water preserved by both preservation

methods (phosphoric add only and phosphoric acid plus ferrous sulfate), as

well as a 1 L deionlzed water blank, were extracted as described by U.S.

EPA Method 604 (2) (a standard separatory funnel extraction). The extracts

were then analyzed by gas chromatography/mass spectrometry (GC/MS). No

compounds were Identified In extracts from either aliquot of sample water.

The 4-aminoant1pyr1ne test for total phenols content was reperformed on the

aqueous residues following extraction. Total phenols were found to be less

than 0 on these aqueous residues. This Indicated to us that the

compound(s) responsible for the Initial positive total phenols value were

either extracted or had decomposed during extraction.

At this point, the supply of sample water was exhausted. Additional

water was collected by EnSafe personnel and shipped to RTI by Federal

Express. Since experimental findings to this point had Indicated that the

preservation methods had had no effect, no preservatives were added. The

sample water was kept cold ("Blue Ice") during shipping. The 4-aminoanti-

pyrine test was performed on this fresh water sample immediately on

receipt. Total phenols were found to be 53 ppb. To verify that sulfides

were not present and producing false positive results for total phenols, an

aliquot of sample water was analyzed for sulfides by Method 9030, Office of

Solid Wastes (3). This test produced a negative result and indicated that

sulfides were not responsible for the positive 4-aminoant1pyr1ne test.

Next, a sorbent accumulation column extraction was chosen as an alter-

native to separatory funnel, 11qu1d-Hqu1d extraction. In this technique,

a large volume (10-20 liters) of sample water is passed through a sorbent

material, such as XAD-4 resin, contained in an open chromatography column.

Organics in the water sample are adsorbed on the XAD resin. The resin is

then eluted with methanol, followed by methylene chloride, and the organic

solvents concentrated to about 1 mL. This method provides for a large

concentration factor because of the large sample volume that can be used

and therefore provides lower detection limits than can be obtained by a

separatory funnel extraction, 1n which only 1 L is processed.

For the sorbent accumulation column extraction, 15 L of sample water

was combined in a glass carboy, and the pH adjusted to 8. Copious

quantities of a whitish-brown precipitate were observed when the pH was

adjusted to 8. This precipitate was believed to be some insoluble iron

compound. The water was passed through an XAD-4 column, which contained 10

mL apparent voluim (after settling) of XAD-4 resin that had been precleaned

by Soxhlet extraction first with ethyl acetate, then with methanol. It

should be noted that the elutlon of the pH 8 water through the XAD column

was very slow due to the precipitate 1n the water. The sample water was

collected in a second glass carboy after passing through the column. The

pH of the water was then adjusted to 2, and the water passed through a

second XAD-4 column. Both of these XAD columns, plus a "blank" XAD column

through which no water had been passed, were then eluted with 12 mL of

methanol, followed by 15 mL of methylene chloride. The organic extract

obtained from the pH 8 XAD column was noted to contain two li q u i d layers

(presumably an aqueous layer on top of an organic layer), both of which

were dark brown in color. The extract from the pH 2 column was single

phase and dark brown In color, while the extract from the "blank" column

was single phase and light yellow in color. Both phases from the pH 8

column, as well as the other two extracts were analyzed by gas chromato-

graphy/flame ionizatlon detection (GC/FID) and by reversed phase high

performance liquid chromatography (RP/HPLC) with UV detection. The chroma-

tograms obtained from the sample extracts were quite complex and are shown,

together with the chromatograms from the "blank" extract, in Figures 1-8.

Because of the complexity of these extracts, it was decided to fractio-

nate them via silica column chromatography prior to GC/MS analysis. Each

silica column contained 5.0 g of cleaned and deactivated silica. The

sample extracts were solvent-exchanged to pentane and then applied (1 mL

volume) to the top of the column. The column was then eluted with 10 mL

T"

N B — —CD

O

rxjfu" r^^/M ro'

ii i IK IK • •H«tm'iimi«» 11, • in it •nunn ••!•! IIIIIMII

Time ( m i n u t e s )

F igure 2 . GC/FID Chromatogram of Bottom Layer of pH 8 Sorbent AccumulatorColumn Extract at 1500 mV full scale. 30 n DB-1 fused silicacapillary column, temperature programmed from 50°C to 250°C at5°C/min.

i T T1 T

00

T - '

\r. IT. «rnjtni .TI . (Di~ f '•

Time ( m i n u t e s )

Figure 3. GC/FID Chromatogram of Top Layer of pH 8 Sorbent AccumulatorColumn Extract. 500 mV full scale, 30 m DB-1 fused silicacapillary column, temperature programmed from 50°C to 250°C at5°C/min.

Time fmlnutes)

Figure 4. GC/F1D Thromatogram of pH 2 Sorhent Accumulator Column Extract500 mV f u l l scale, 30 m DB-1 fused s i l i c a c a p i l l a r y column,temperature programmed from 50°C to 250°C at 5°C,min.

J900.800

.ecu

10flinutes

ô

Figure 5. HPI.C Chromatogram of Blank Sorbent Accumulator Column Extract at1900 mV full scale, UV detection at 254 nm, Zorbax C8 Column (2ocm x 4.6 mm ID), 10% MeCN/^O to 90% MeCN/H.,0 over 20 nin, 1.5

mL/min flow rate.

1900.000

5.000

-16.000• JtfW-1

-i »—10 15Minutes

Figure 6. HPLC Chromatogram of Bottom Layer of pH 8 Sorbent AccumulatorColumn Extract at 1900 mV full scale, UV detection at 254 nm,Zorbax C8 Column (25 cm x 4.6 mm ID), 10% MeCN/H 0 over 20 min,1.5 mL/min flow rate.

-10.000

10

Figure 7. HPLC Chromatogram of Top Layer of pH 8 Sorbent Accumulator ColumnExtract at 1900 mV full scale, UV detection at 254 nm Zorhax C8Column (25 cm x 4.6 mm ID), 10% MeCN/HgO to 90% MeCN/HgO over 20min, 1.5 mL/min flow rate.

-te.eee10Minutes

Figure 8. HPLC Chromatogram of pH 2 Sorbent Accumulator Column Extract at1900 mV full scale. UV detection at 254 nm, Zorbax C8 Column (25cm x 4.6 mm ID),mL/min flow rate.

10% MeCN/H 0 to 90% MeCN/H 0 over 20 min, 1.5

pentane (Fraction I), 50 mL of methylene chloride/pentane (20:80) (Fraction

II), and 100 mL of methanol/methylene chloride (6:94) (Fraction III). Each

fraction was concentrated to exactly 1 mL by rotary evaporation and

nitrogen blowdown. All fractions were analyzed by GC/FIO and RP HPLC/UV.

The resulting chromatograms are shown 1n Figures 9-32 and show varying

degrees of complexity.

Based on the appearance of these chromatograms, It was decided to

submit Fractions II and III of all extracts except the blank for GC/MS

analysis. Fraction III of the pH 2 extract was submitted for analysis by

HPLC/MS in an attempt to Identify the large, tailing, early-elutlng peak

present in the HPLC/UV Chromatogram of that extract.

3.0 CONCLUSIONS

Examination of the GC/MS data revealed a large number of compounds

found in each fraction. Table 1 lists those compounds found at the highest

apparent concentrations. Precise quantHatlon of the level found could not

be determined 1n those extractions where the target compound(s) were not

known In advance. Of particular note 1n Table 1 Is the rather large number

of alkyl phenols that were found. We believe that this large number of

different phenols that were all present at very low levels «2 ppb) in the

water sample accounts for the failure to detect them during the standard

separatory funnel extraction, 1n which only one liter of sample was extrac-

ted. It also accounts for the 10-50 ppb value found with the 4-aminoanti-

pyrine test for phenols based on the sum of the masses of the many

different phenols present. These alkyl phenols were found in both

Fractions II and II of the silica cleanup procedure and also in both layers

of the pH 8 extract and In the pH 2 extract.

14

10.00 20.00 30.00 40.00

F i g u r e 9 . Gt.'FlD Chromatograrr of Fraction 1 of Blank Sorbent AccumulatorColumn Extract at 100 mV f u l l scale, obtained using a 30 m DB-1fused silica capillary column, temperature programmed from 50PC

to 250IJC at 5°C/min.

\lL

,0.00 20.00 30.00 40.00

Figure 10. GC/FID Chromatogram of Fraction 2 of Blank Sorbent AccumulatorColumn Extract at 100 mV full scale, obtained using a 30 IB DB-1fused silica capillary column, temperature programmed from 50°Cto 250°C at 5°C/min.

10.00 20.00 30.00 40.00

Figure 11. GC/FID Chrc.matogram of Fraction 3 of Blank Sorbent Accumulator-Column Extract at 100 mV full scale, obtained using a 30 m DB-1fused silica capillary column, temperature programmed from 50°Cto 250°C at 5°C/min.

T

10.00 20.001

30.00 40.00

Figure 12. GC/FID Chnm.at ogram of Fraction 1 of Bottom Layer of pH 8 SorbentAccumulator Column Extract at 100 mV full scale, obtained using a30 ni DB-1 fused s i l i c a capillary column, temperature programmedfrom 50°C to 250°C at 5°C/min.

20.00 30.00 40.00

Figure 13. GC/FID Chronirit ogram of Fraction 2 of Bottom Layer of pH 8 SorbentAccumulator Column Extract at 100 mV full scale, obtained using a30 m DB-1 fused silica capillary column, temperature programmedfrom 50JC to 250JC at 5uC,min.

NJO

10.00 20.00 30.00 4 PI

Figure 14 GC/FID Chromatogran, of Fraction 3 of Bottom Layer of pH 8 SorhrntAccumulator Column Extract at 100 mV full scale, obtained using a30 IP DB-1 fused silica capillary column, temperature programmedfrom 50°C to 250°C at 5°C/min.

toO

10.00 20.00 30.00 4 Pi

Figure 14. GC/FID Chromatogram of Fraction 3 of Bottom Layer of pH 8 SorbentAccumulator Column Extract at 100 mV full scale, obtained using a30 m DB-1 fused silica capillary column, temperature programmed

from 50°C to 250°C at 5°C/min.

10.00 20.00 30.00 40.00

Figure 15 GC/FID Chromatogram of Fraction 1 of Top Layer of pH 8 Sorbent' Accumulator Column Extract at 100 mV full scale, obtained using a

30 m DB-1 fused s i l i c a capillary column, temperature programmed

from 50UC to 250°C at 5°C/min.

JuJLjL

1J 0 . 0 0 20.00 30.00 40.00

F i g u r e 1 C . G C / F I D C h r o m a t o g r a m of Frac t ion 2 of Top Layer of pH 8 SorbentA c c u m u l a t o r C o l u m n Ex t rac t at 100 mV f u l l scale , obtained us ing a30 m DB-1 f u s e d s i l i c a c a p i l l a r y c o l u m n , t e m p e r a t u r e p r o g r a m m e df r o m 50°C to 250°C at 5 ° C / m i n .

10.00 20.00 30.00 40.00

Figure 17 GC/FID Chromatogram of Fraction 3 of Top Layer of pH 8 SorbentAccumulator Column Extract at 100 mV full scale, obtained using a30 n DB-1 fused silica capillary column, temperature programmedfrom 500C to 250"C at 5°C/min.

X...A-

0.00 20.00 30.00 40.00

Figure 18. GC/FID Chromatogram of Fraction 1 of PH 2 Sorbent AccumulatorR Column Extract at 100 mV full scale, obtained using a 30 m DB-1

fused silica capillary column, temperature programmed from 50 L

to 250°C at 5°C/min.

*w

110.00 20.00 30.00 40.00

Figure 19. GC/FID Chromatogram of Fraction 2 of pH 2 Sorbent AccumulatorColumn Extract at 100 mV full scale, obtained using a 30 m DB-1fused silica capillary column, temperature programmed from 50°Cto 250°C at 5°C/min.

110.00 20.00 30.00 40.00

Figure 20. GC/FID Chromatogram of Fraction 3 of pH 2 Sorbent AccumulatorColumn Extract at 500 mV f u l l scale, obtained using a 30 m DB-1fused silica capillary column, temperature programmed from 50°Cto 250°C at 5°C/min.

f«e.600

16 36

Figure 21. HPLC Chromatogram of Traction 1 of Blank Sorbent AccuirulatorColumn Extract at 525 mV full scale, UV detection at 254 nm,Zorbax C Column (25 cm x 4.6 mm ID), 10% MeCN/H 0 to 90%MeCN/H 0 over 20 m i n , 1.5 mL/min flow rate.

recc

if ,?oni^tn

Figure 22. HPI.C Chromatogram of Fraction 2 of Blank Sorbent AccumulatorColumn Extract at 525 mV full scale, UV detection at 254 nm,Zorbrix C,, Column (25 cm x 4 .6 mm ID), 10% MeCK/H^O to 90%C ColumnMeCN/H 0 over 20 min. 1.5 mL/min flow rate

10 JO 40

Figure 23. HPLC ChromatogramColumn Extract atZorbax C ColumnMeCN/H 0 over 20

of Fraction525 »V full(25 cm x 4.6

3 of Blank Sorbrnt Accumulatorcsale, UV detection at 254 nm,mm ID), 10% MeCN/HmO to 90%10% MeCN/H 0

m i n , 1.5 ml./min flow rate.

1II

, X>0

la is LO zfj M J5 40

Figure 24 HPLC Chromatogram of Fraction 1 of Bottom Layer of pH 8 SorbentAccumulator Column Extract at 525 mV full scale, UV detection at254 nm, Zorbax C Column (25 cm x 4 .6 mm ID), 10% MeC.N/^O to 90%

MeCN/H 0 over 20 mi n , 1.5 ml./min flow rate.

15 COn 1 r,o l r £ 40

Figure 25. HPI.C Chromatogram of Traction 2 of Bottom Layer of pH 8 SorbentAccumulator Column Extract at 525 mV full scale, I'V detection254 nm, Zorhax C Column (25 cm x 4.6 mm ID).MeCN/H 0 over 20 min, 1 m l - m i n f l o w r a t e .

at10% MeCN H 0 to 90%

10 lfj

254 nm. Zoibax C Column (25 cm x 4.fi mmMeCN'H 0 over 208min, 1.5 mL/min flow rate.

10% MeCN/H 0 to 90%

•rvooo

Figure 27. HPLC Chromatogram of T r a c t i o n 1 of Top Layer of pH 8 ScrbentAccurrulator Column E x t r a c t at 525 mV full scale, I'V detection at2">4 nm. 7orbax Cn Column (25 cm x 4.6 mm ID),7oibax C Column (25 cm x 4MeCN.-'H 0 over 20 m i n , 1 5 ml./IT, in flow rate.

10% M e f N / H O to 90%

.000

LJ-t-

10 Jit' 35 40

Figure 28. HPLC Chromatogram of Fraction 2 of Top Layer of pH 8 SorbentAccumulator Column Extract at 525 mV full scale, UV detection at254 nm, Zorbax C Column (25 cm x 4.6 mm ID), 10% MeCN/H 0 to 90%MeCN/H 0 over 20 min, 1.5 ml./min flow rate.

-5.000

iO 15 .1-0IV. r>JI»J 40

Figure 29. HPLC Chromatogram of Fraction 3 of Top Layer of pH 8 SorbentAccumulator Column Extract at 525 mV full scale, UV detection at254 nm, Zorbax C Column (25 cir. x 4.6 mm ID), 10% MeCN/H 0 to 90%MeCN/H 0 over 20 min. 1.5 mL/'min flow rate.

j

roi.oco !

iJ-1.000 I

X

Figure 30. HPLC Chromatogram of Fraction 1 of pH 2 Sorbent AccumulatorColumn Extract at 525 mV full scale. UV detection at 254 nm,Zorbax C Column (25 cm x 4.6 mm ID), 10% MeCN/H 0 to 90%

MeCN/'H 0 over 20 min, 1.5 mL/min flow rate.

* 1-5.000

\

0 J»0 40

Figure 31. HPLC Chromdtogram of Fraction 2 of pH 2 Sorbent AccumulatorColumn Extract at 525 mV full scale, UV detection at 254 nm,Zorbax C Column (25 cm x 4.6 mm ID). 10% MeC.N/H 0 to 90%MeCN'K 0 over 20 min. 1.5 mL/min flow rate.

Figure 32. HPLC Chromat cigram of Traction 3 of pH 2 Sorberit AccumulatorColumn Extract at 525 mV f u l l scale, UV detection at 254 nm ,Zorbax C Column (25 cm x 4.6 mm ID),MeCN/H 0 over 20 min,

10% MeCN/H 0 to 90%1.5 ml./'min flow rate.

TABLE 1. COMPOUNDS/COMPOUND CLASSES IDENTIFIED BY GC/MS

2 Methoxy phenols

5-7 C3-Alkyl phenols

5-6 C4~Alkyl phenols

1-2 Cs-Alkyl phenols

2 Ethyl methyl phenols

Dimethyl phenols

Trimethyl phenols

CjH\QQ ketone

Dimethyl thiolndene

Benzole acid

n-Decane

Isopropyl benzoate

Alkyl esters,

4-(2,5-Xylyl) butanoic acid

Methyl myristate

Methyl palmitate

Methyl stearate

n-Undecane

Dlmethylbenzofuran

Alkyl benzenes

3'*

HPLC/MS analysis of the pH 2, Fraction III extract revealed a very

complex sample. The mass spectra of the early-elutlng, tailing peak

exhibited a peak at every mass unit In the range of 100-320 amu, Indicating

high matrix interferences and preventing identification or postulation of

compound(s) or class(es). Further fractlonation work would be necessary

for compound identification to be successful. Since satisfactory GC/MS

results were obtained, no further HPLC/MS work was pursued.

4.0 RECOMMENDATIONS

The procedure employed worked well for the isolation and identification

of the various alkyl phenols. Based on final results, two changes are

recommended to reduce analysis time and costs and to Improve the overall

detection limit. First, since the alkyl phenols were partially extracted

during the pH 8 sorbent column extraction, we recommend that the sample be

extracted only once at a pH of 2. This will reduce the time required for

both extraction and GC/MS analysis and w i l l also lower detection limits by

Inclusion of target compounds 1n one extract. This will also avoid genera-

tion of the insoluble precipitate, which slowed sample elution through the

XAD column. Second, since the alkyl phenols eluted 1n both Fractions II

and III of the silica column cleanup, we recommend that these fractions be

combined prior to GC/MS analysis. Again, this will reduce GC/MS costs and

lower detection limits by concentrating all compounds in one fraction.

Proposed protocols for the extraction and cleanup procedures are provided

1n Appendix A.

5.0 REFERENCES

1. Standard Methods for^the Examination of Water and Wastewater, Fifteenth

and Sixteenth Editions, American Public Health Association, 1015

Fifteenth Street, N.W., Washington, DC 20005, 1985.

2. Test Methods: Methods for Organic Chemical Analysis of Municipal and

Industrial Wastewater, EPA-600/4-8^-057, Environmental Monitoring and

Support Laboratory, Office of Research and Development, U.S. Environ-

mental Protection Agency, Cincinnati, OH, 1982.

3- Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, 2nd

Edition, SW-846, Office of Solid Waste and Emergency Response, U.S.

Environmental Protection Agency, Washington, DC, 1985.

APPENDIX A

PROTOCOLS FOR ANALYSIS

PROTOCOL A: SORBENT ACCUMULATOR COLUMN (SC) EXTRACTION

1.0 INTRODUCTION

1.1 Principle of the Technique

This method describes the determination of acid extractable organic

compounds in drinking water samples. Resin materials are used to adsorb

and concentrate organic compounds from 15.0 L water samples. A nominal 0.8

ppb detection l i m i i 1s achieved using this size sample. Although the

method has seen developed specifically for drinking water, other water

types, such as some surface waters may also be processed depending upon

desired samole siz\ organic content, and turbidity. The sample pH is

adjusted to 2 and the sample passed through a 10 mL Amberlite XAD-4 macro-

reticular resin column. Excess water is aspirated off the column and

adsorbed organics are eluted using 12 mL of methanol followed by 200 mL

methylene chloride. The eluants are combined, then evaporated to a volume

of 4 mL using a Kuderna-Danish evaporator and further concentrated to 0.5

mL by nitrogen blowdown using a modified micro-Snyder column. Neutral and

acidic organics are analyzed using this procedure.

1.2 Detection Limit

If the detection limit for GC/MS analysis 1s estimated at 10 ng total

material for extractable organics, and samples are concentrated from an

original volume of 15.0 L to a final extract volume of 1.0 mL, the overall

nominal detection limit for this procedure would be 0.8 ppb for a 1 /*L

injection.

Detection limits may be decreased by further increasing sample size to

50 to 100 L. If this approach 1s taken, column accumulation should be

performed in the field due to the prohibitive cost of shipping the samples.

Breakthrough characteristics using large water volumes are unknown and

recoveries may be significantly decreased.

1.3 Interferences

Interferences may be a problem with complex water samples. Extracts

from such samples may be fractionated via s i l i c a column as described in a

separate protocol. A procedural blank must be run prior to processing any

samples to assure that contamination from resin materials, solvents,

reagents, glassware, and other sources is low.

2.0 APPARATUS AND REAGENTSThe following materials are required for processing a set of eight

samples (7 water samples and a procedural blank). Every set of samples

must contain a procedural blank. The extraction/concentration procedure

w i l l require two working days for a set of eight samples.

(1) Eight glass chromatography column ~1 cm i.d., x 22 cm with ateflon stopcock and a 24/40 ground glass joint at the top. Each

chromatography column should have an adaptor (Figure 1) forsiphoning the samples onto the resin.

(2) One vacuum aspirator for pulling water through the resin column.(3) One 10 mL graduated pipette with the tip cut off for measuring and

transferring resin material.

(4) Eight 500 mL Erylenmeyer flasks.

(5) Glass wool (-50 mL) for resin and drying columns. Glass woolshould be precleaned by Soxhlet extracting overnight with

methylene chloride.

(6) GKV..S b o i l i n g beads.(7) On. Soxhlet extractor fur cleaning resin material and glass wool.

A minimum ?00 mL capacity Is required. Resins can be processed in

la.je batches and stored; therefore a 500 mL to 1000 mL size would

be preferred.

(8) Eight Kuderna-Danish apparatus. The apparatus consists of:

(a) Three-ball macro-Snyder columns [Kontes 0K503000] - 300 mm

length with 24/40 joints.

(b) Evaporative flask {Kontes 0K57000?] - 250 ml with 24/40 top

joint and 19/22 lower joint.

(c) Concentrator tube [Kontes rfK570000] - 4 mL graduated, with

19/22 joint. Attach to evaporative flask with springs

[Kontes K503000-0232]. Ground glass stopper (size 19/22

joint) is used to prevent evaporation of extracts.

After rinsing three times with solvent, the K-D apparatus can be

used for both the bai:e and acid extracts of the same water sample.

(9) Eight modified micro-Snyder columns [Kontes 0K569251] with 19/22ground glass joint.

(10) One heated water bath or steam bath with concentric ring covercapable of temperature control. Bath should be located in a hood.

(11) One pH meter.

(12) One manifold with temperature controlled water bath or aluminumheating block for nitrogen blowdown. A manifold with eight spaces

is preferred but not essential.

(13) Pasteur pipettes.(14) Eight 1 dram glass vials with screw caps (Supelco 2-3213) and

Teflon-lined rubber septa (Supelco 2-3216).

(15) One gas chromatography column; for example, 0.32 mm i.d., x 30 mDB-1 fused s i l i c a capillary column.

(16) Materials and Regents(a) Redistilled methanol (Burdick and Jackson, pesticide analysis

grade).

(b) Redistilled ethyl acetate (Burdick and Jackson, pesticide

analysis grade).

(c) Methylene chloride (Burdick and Jackson, pesticide analysisgrade).

(d) 160 mL Amberlite XAD-4 macroreticular resin (Rohm and Haas,

Philadelphia, Pennsylvania).

(e) 100 mL 6N H2S04.

(f) 100 mL 6N NaOH.

3.0 PROCEDURE

3.1 Sample Collection, Preservation and Handl HKJ

3.1.1 Sample Preservation

The samples must be iced or refrigerated from the time of collectionuntil extraction.

3.1.2 r imple Handling

All samples should be analyzed w i t h i n 14 days of collection.

3.1.3 flipping

Samples should be shipped on ice directly to the laboratory by an

appropriate air carrier (e.g., Federal Express) in well insulated and

packed cartons.

3.2 Sample Workup

3.2.1 Cleaning of Glassware

A l l glassware to be used Including sample bottles should be washed with

Isocleanfl (diluted 1 to 40 with d i s t i l l e d water), rinsed with d i s t i l l e d

deioniml water and heated to 400°C 1n a glassware oven.

3.2.2 Preparation of Resin Material

Amberlite XAD-4 resin material is cleaned prior to preparing the resin

columns. The minimum amount of resin that should be prepared is 160 mL,

which is the amount needed to process eight water samples. The following

instructions describe procedures based on 160 mL of resin material;

however, it is possible to prepare larger batches and store the prepared

resin in methanol in a sealed glass jar. If a larger volume of resin is

prepared, reagent volumes should be adjusted proportionately.

Place the resin material In a 500 mL Erylenmeyer flask. Make a slurry

of the resin with 250 mL of d i s t i l l e d water. Stir the resin gently to

avoid fracturing beads. After allowing the resin to settle for 30 seconds,

remove fines by decantatlon. Repeat the slurry-decantatlon procedure three

times. Then rinse the resin three times with 250 mL portions of methanol.

Place the resin In a Soxhlet extractor and extract with pesticide analysis

grade ethyl acetate for eight to sixteen hours (cycle time -10 minutes).

Then extract the resin with pesticide analysis grade methanol for an

additional eight to sixteen hours (cycle time: ~10 minutes). Store

cleaned resin in a sealed glass jar in methanol with a Teflon-lined screw

cap.

3.2.3 Sample Extraction

(1) Prepare resin columns by placing a small glass wool (pre-

extracted) plug in the bottom of the chromatography column. Add

10 mL of methanol to the column. Pipette 10 mL (allow the resin

to settle in the pipette before measuring) of cleaned Amberlite

XAD-4 synthetic resin into the columns using a 10 mL graduated

pipette with the tip cut off. Allow the resin to settle in the

column, then drain the methanol to the top of the resin bed.

f i n a l l y rinse the column with 10 bed volumes (100 mL) of d i s t i l l e d

water.

(2) Adjust the pH of the aqueous sample to 2 using 6N H2S04 or 6NNaOH.

(3) Attach siphoning adaptor (Figure 1) between sample container andresin column. Attach a piece of tygon tubing between the end of

the chromatography column and a water aspirator. To start sample

flowing through the resin, open column stopcock and turn on

aspirator. This should create sufficient suction to pull thewater sample through the siphon apparatus onto the resin column.

Remove the tycjon tubing.

(4) Using the stopcoc!. at the base of the column, adjust flow throughthe column to ~10 mL/minute. Sample may be discarded after elu-

tion through the column.(5) After the sample has passed through the column, remove the siphon

adaptor from the column. If any sample remains 1n the containers,transfer It to the column and pass remaining sample through the

resin bed.

(6) Once all sample has passed through the column, attach a piece of

tygon tubing between the end of the column and a water aspirator.Aspirate any remaining water off the column (aspirate for 2 to 3minutes). It Is important to remove all water during this proce-dure.

(7) Add 12 mL of methanol to the dried resin bed. Stopper the column

and shake to remove any air bubbles. Equilibrate the column for

10 minutes.(8) Rinse the containers for each sample with a total of 200 mL

methylene chloride. Pour the methylene chloride rinse into the

separatory funnel.

(9) Drain all of the methanol from the resin bed. Collect the eluate

in a 500 mL Erlenmeyer flask.

(10) Add ~15 mL methylene chloride to the resin bed, allow air bubbles

to escape. Place a glass wool plug on the top of the resin bed.

(11) Collect ~5 mL of methylene chloride from the separatory funnel,

then return to the separatory funnel. This removes any entrained

water from the stopcock and connector joint.

ryjoo tubirvg to

Figure 1. Accumulator coli.mn with siphon adaptor.

(12) Elute the column with the remaining methylene chloride. Stopelutfng when the methylene chloride reaches the top of the resinbed. Do not elute any water through the resin. Collect eluant inthe 500 mL Erlenmeyer flask containing the methanol rinse. Eluant

may be stored at 0°C after this operation.

(13) Transfer the eluant to a 500 mL K-D flask equipped with a 10 mL

concentrator tube containing several glass boiling beads.

Precallbrate the concentrator tube to compensate for the volume of

the glass boiling beads. Rinse the bottle with three 5 mL

portions of methylene chloride and transfer three rinses to the K-D flask. Attach the three-ball macro-Snyder column. Place the K-

D apparatus on a warm water bath with the concentrator tube

partially Immersed 1n water or on a steam bath. Adjust the tem-perature of the water bath such that evaporating solvent causes

the balls 1n the Snyder column to chatter actively but does not

flood the chambers of the column. Under these conditions evapora-

tion of 500 mL of methylene chloride to 2 mL should take 15 to 20

minutes.(14) When the liquid has reached an apparent volume of 2 mL remove the

K-D apparatus from the heat and allow to cool. Remove the Snyder

column. With 1 to 2 mL of solvent, rinse the evaporation flask

and its lower joint Into the concentrator tube. The final volume

in the concentrator tube should be approximately 4 mL.

(15) Attach a modified micro-Snyder column to the K-D receiver.

Connect a transfer pipette to the nitrogen manifold using Teflon

tubing. Placea the pipette in the modified micro-Snyder column

above the solvent level. Gently blow a stream of nitrogen above

the solvent surface until the volume is reduced to approximately 1

mL (CAUTION: contamination can occur if tygon tubing is used to

connect the manifold to the nitrogen source). Rinse the sides of

the concentrator tube with approximately 0.5 mL of solvent.

Reduce volume of eluant to 0.5 mL using nitrogen blowdown. Record

final volume.

49

PROTOCOL B: CLEANUP/FRACTIONATION

1.0 INTRODUCTION

This pr .tocol describes a procedure for fractionating extracts identi-

fied as containing high levels of chromatographable organics. These highlevels of c.ganlc substances would prevent the Identification and quantita-tion of analytes at the levels specified for the overall methods.

The fractionatlon 1s performed using a 5 g silica gel open column. The

silica gel is activated at 150°C and deactivated with 14 mL of water per

100 g of s i l i c a gel. The sample Is then fractionated Into three fractions

consisting of pentane (20 mL), 20% methylene chloride In pentane (50 mL)

and 6% methanol In methylene chloride (100 mL). The second two fractionsmay be recombined for GC/MS/DS analysis.2.0 APPARATUS AND REAGENTS

The following materials are required for processing a set of eight

sample extracts. The fractionation of eight sample extracts w i l l require

one and a half working days including solvent evaporations.

(1) Eight chromatography columns, 220 mm x 10 mm i.d. with Teflon

stopcocks and 24/40 ground glass joints at the top.(2) Eight 250 mL dropping funnels with 24/40 ground glass joints and

Teflon stopcocks.(3) Eight glass funnels to fit dropping funnels.

(4) Pre-extracted glass wool (~50 mL) for columns. Glass wool should

be precleaned by Soxhlet extracting overnight with methylene

chloride.

(5) One Soxhlet extractor (50 mL capacity minimum) for extracting

glass wool.

(6) Eight Kuderna-Danish apparatus. Each apparatus consists of

(a) Three-ball macro-Snyder columns (Kontes 0K503000) - 300 mm

length with 24/40 joints,

(b) Evaporative flask (Kontes 0K570001) - 250 mL with 24/40 top

joint and 19/22 lower joint,(c) Concentrator tube (Kontes 0K570000) - 4 mL graduated, with

19/22 joint. Attach to evaporative flask with springs

(Kontes IK50300-0232). Ground glass stopper (size 19/22joint) 1s used to prevent evaporation of extracts.

(7) Sixteen modified micro-Snyder columns (Kontes #K569251) with 19/22

joint.(8) Sixteen concentrator tubes (Kontes #K570050-25) - 25 mL graduated,

with 19/22 joints.(9) Boiling chips - Hengar granules are ground with a mortar and

pestle and sieved to obtain a 60/80 mesh. These granules arecleaned by extracting ~5 g three times with ~150 mL methylene

chloride.(10) Water bath - Heated, with concentric ring cover, capable of tempe-

rature control (+2°C). The bath should be used in a hood.

(11) Or.o manifold with a temperature controlled water bath for nitrogenbmwdown. A manifold with eight spaces 1s preferred but not

essential.

(12) Eight glass vials (1 dram) with 13 mm screw caps (Supelco 2-3213)

and Teflon lined rubber septa (Supelco 2-3216).

(13) One gas chromatography column (180 cm x 2 mm i.d.) packed with 3%OV-1 on Chromosorb W HP (80/100 mesh) or equivalent.

(14) One gas chromatograph with flame ionization detector.

(15) One laboratory oven.

(16) Materials and Reagents

(a) 1.5 L methylene chloride (Burdick and Jackson, distilled in

glass).(b) 0.5 L pentane (Burdick and Jackson, distilled in glass).

(c) 50 mL methanol (Burdick and Jackson, distilled in glass).

(d) 100 g silica gel 100/120 mesh (Fisher reagent grade).

(e) 1 mL column performance standard (Table 1).

52

TABLE 1. COLUMN PERFORMANCE STANDARD

CompoundConcentration

mg/10 mLa

Naphthalene

Anthracene

10

20

aMethylene chloride solvent.

(f) Reagent water - reagent water 1s defined as a water sourcewhich does not produce a background Interference at the l i m i tof detection. A water purification system (MilUpore Super-Qor equivalent) may be used to generate reagent water.

3.0 PREPARATION FOR FRACTIONATION(1) Cleaning of GlasswareAll glassware to be used should be washed with Isoclean (diluted 1 to

40 with distilled water), rinsed with distilled deionlzed water and heatedto 400°C in a glassware oven for a'minimum of 4 hours.

(2) Activation of the Silica GelSilica gel (100 g) 1s placed 1n a wide mouth jar in an oven at 150°C

for a minimum of 14 hr. Immediately prior to use, cap the jar and place ina desiccator to cool. When cool, add 14 mL of reagent water and agitateuntil evenly distributed. Allow to equilibrate for at least 2 hrs.

(3) Cleaning Glass WoolGlass wool is r'eaned by placing in the extraction chamber of a Soxhlet

extractor and extracting overnight with pesticide grade methylene chloride.The glass wool 1s placed 1n a wide mouth jar and the solvent removed eitherunder nitrogen stream or by warming. Cap and store until needed.4.0 SAMPLE FRACTIONATION

4.1 Determination of Silica Gel Activity

(1) A chromatography column containing 5.00 + 0.05 g of silica gel isprepared by layering the silica gel on top of a plug of glass wooland tapping the column gently to settle the sorbent.

(2) Dilute 0.5 mL of the column performance standard (Table 1) to 10mL with pentane and mix gently.

53

(3) Transfer the diluted column performance mixture to the silica gelcolumn and allow the solution to elute through the column u n t i lthe solvent reaches the top of the silica gel. Collect thiseluate.

(4) Elute the column with an additional 10 mL of pentane and collectthis eluate as above (3) 1n the same container. This should be~15 mL.

(5) Dilute 0.5 mL of column performance standard with 15 mL of pentaneand mix.

(6) Inject the column eluate (4) on the 180 cm x 2 mm i.d. 3% OV-1 onChromosorb W HP packed GC column. Compare peak areas (or heights)directly against those obtained from an equal volume injection ofthe diluted performance standard (5).

(7) If the naphthalene recovery Is 70% and theanthracene recovery Is ^50%, Increase the amount of silica gel by0.05 g and repeat steps 1-6. If the naphthalene recovery 1s >70%and the anthracene recovery is

(5) The redlluted extract 1s transferred to a column and the solventeluted until 1t reaches the surface of the silica gel. Collectthis eluate 1n a 25 mL K-D concentrator tube.

(6) Rinse the extract container 1n 2 mL portions with a total of 10.0mL of pentane transferring each portion to the column. Elute thissolvent until the liquid level reaches the sorbent and close thestopcock. Collect this eluate with that from step 5 and label asFraction I. The volume of eluate should be ~15 mL.

(7) Transfer the eluate to a 250 mL K-D evaporator flask fitted with a4 mL graduated concentrator tube.

(8) Measure 50 mL of 20% methylene chloride, 80% pentane (v/v). Rinsethe sample extract container (from step 2) three times with 2 mLportions of this solvent and transfer to the column. Add remain-ing solvent to column using a dropping funnel. Elute the columnuntil the solvent reaches the top of the silica gel. Collect in a250 mL K-D flask equipped with 4 mL concentrator tube containingseveral boiling chips. The concentrator tube should beprecalibrated to compensate for the volume of the boiling chips.

(9) Measure 100 mL of 6% methanol 1n methylene chloride. Rinse thesample extract container three times with 2 mL portions of thissolvent and transfer to the column. Add remaining solvent to thecolumn using a dropping funnel. Elute the column until thesolvent reaches the top of the silica gel. Label the combinedeluates from steps 8 and 9 as Fraction II.

(10) Evaporate Fraction I to 1.0 mL by nitrogen blowdown with a modi-fied micro-Snyder column as described in step 1.

(11) Evaporate the eluate labeled Fraction II. Attach the three-ballmacro-Snyder column. Place the K-D apparatus on a warm water bathwith the concentrator tube partially immersed In water or on asteam bath. Adjust the temperature of the water bath such thatevaporating solvent causes the balls In the Snyder column tochatter actively but does not flood the chambers of the column.Under these conditions evaporation of 150 mL of solvent to 2 mLshould take 10 to 15 minutes.

55

(12) When the liquid has reached an apparent volume of 2 mL remove theK-D apparatus from the heat and allow to cool. Remove the Snydercolumn. With 1 to 2 mL of solvent, rinse the evaporation flaskand Its lower joint Into the concentrator tube. The .final volume1n the concentrator tube should be approximately 4 mL.

(13) Attach a modified micro-Snyder column to the concentrator tube andconcentrate to 1.0 mL as described in step 2.