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The Analysis of OpticalBrightening Agents inPaper Samples Using LiquidChromatography with High-Resolution Mass SpectrometryFarzad Shadkami a , Robert Helleur a & Bruce B.Sithole ba Analytical Chemistry Group, Department ofChemistry, Memorial University of Newfoundland, St.John's, Newfoundland, Canadab Pulp and Paper Research Institute of Canada,Pointe-Claire, Quebec, Canada

Available online: 03 Mar 2011

To cite this article: Farzad Shadkami, Robert Helleur & Bruce B. Sithole (2011): TheAnalysis of Optical Brightening Agents in Paper Samples Using Liquid Chromatographywith High-Resolution Mass Spectrometry, Journal of Wood Chemistry and Technology,31:1, 42-57

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Journal of Wood Chemistry and Technology, 31: 42–57, 2011

Copyright © Taylor & Francis Group, LLC

ISSN 0277-3813 print / 1532-2319 online

DOI: 10.1080/02773811003725695

The Analysis of Optical Brightening Agentsin Paper Samples Using Liquid Chromatography

with High-Resolution Mass Spectrometry

Farzad Shadkami,1 Robert Helleur,1 and Bruce B. Sithole2

1Analytical Chemistry Group, Department of Chemistry, Memorial Universityof Newfoundland, St. John’s, Newfoundland, Canada

2Pulp and Paper Research Institute of Canada, Pointe-Claire,Quebec, Canada

Abstract: There is limited information available on the analysis and characterizationof optical brightening agents (OBA) commonly used in the pulp and paper industry. Theavailable literature provides only a few reports on suitable chromatographic methodsof analysis of OBA compounds present in paper products. The choice of suitableextraction solvents for OBA is also debatable. Some advocate using organic solvents,whereas others use water. The extracted OBAs are usually characterized by fluorescencespectroscopy or liquid chromatography with UV detection. These methods are notselective and do not yield information on the chemical structures of the compounds.We have characterized OBA compounds by liquid chromatography with high-resolutionmass spectrometry, specifically liquid chromatography–quadrupole time-of-flight–massspectrometry (LC-QTOF-MS). Preliminary extraction results show that boiling water iseffective for characterizing OBAs from commercial papers. Positive-ion and negative-ion electrospray ionization allowed for observation of straightforward mass spectrafor OBA standards and OBA extracts from paper products. The ESI-MS results forthe most common OBA in the industry, TOBA (a tetrasulfonated stilbene derivative),gave a prominent m/z value of 1075.1797 in the negative-ion mode and 1077.1941in the positive-ion mode corresponding to the [M − 1]− and [M + 1]+ species oftetrasulfonated optical brightening agent in the full acidic form. The LC-MS analysisof OBAs in paper extracts showed that they can exist in both cis and trans forms, animportant parameter for proper quantitative analysis of OBAs. Preliminary experimentsfound that isomerization can actually occur when certain OBAs in solution are exposedto natural light.

Keywords: Optical brightening agents, fluorescent whitening agents, QTOF, LC-QTOF-MS, optical brighteners, fluorescent brightening agents, extraction

Address correspondence to Farzad Shadkami, Department of Chemistry, University ofToronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6. E-mail: shadkami@mun.ca

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Analysis of Optical Brightening Agents 43

INTRODUCTION

Optical brightening agents (OBAs), sometimes called fluorescent whiteningagents (FWAs), are now widely used to achieve a higher standard of whitenessin paper and board used for packaging and writing papers. They influence theoptical properties of the paper directly by means of chemicophysical interac-tions. The compounds absorb light in the UV range, 290–400 nm, and emitvisible blue light, 400–480 nm, thereby enhancing the optical impression ofhigher whiteness and brightness. Consequently, the yellowish tinge of the fibersshifts toward bluish-white. Thus, the degree of whiteness can be increased quitesignificantly, especially if the OBAs are combined with shading dyestuffs.[1]

This in turn leads to sharper contrasts in the printed image and helps to reducetoner consumption. OBAs can be added in the stock preparation, wet end, sizepress, and coater. High- or medium-substantivity compounds are used in stockapplications, whereas medium- or low-substantivity compounds are used in thesize press and coaters. The dosages used are ∼1% in stock applications and1.5% in size press or coaters.[2] Lately, higher dosages, up to 3.5%, have beenused to achieve even higher brightness properties in paper products.

The OBA stilbene derivatives are widely used in the paper industryand include (1) disulfur (or 4,4′-bis[[4-[bis(2-hydroxyethyl)amino]-6-anilino-1,3,5-triazin-2-yl]amino]stilbene-2,2′-disulfonic acid [DOBA]), (2) tetra-sulfur (or tetrasodium 4,4′-bis[[4-[bis(2-hydroxyethyl)amino]-6-(4-sulfonateanilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2′-disulfonate salt [TOBA]), and(3) hexasulfur (or hexasodium 4,4′-bis[[4-[bis(2-hydroxyethyl)amino]-6-(2,5-disulfonate anilino)-1,3,5-triazin-2-yl]amino]stilbene-2,2′-disulfonatesalt [HOBA]) derivatives (Figure 1). Among them, tetrasulfur (TOBA) is themost commonly used.

In addition to the pulp and paper industry, OBAs are widely used in the tex-tile industry and in the manufacture of household detergents. However, there

Figure 1. Original forms (structures) of optical brightening standards as received.Shown are the structures of (1) DADS, (2a) DOBA, (2b) TOBA, and (2c) HOBA.

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44 F. Shadkami, R. Helleur, and B. B. Sithole

are concerns about toxicity and the environmental effects of these products.Governmental agencies such as the U.S. Food and Drug Administration (FDA)require assurance that food packaging will not release OBAs into food. Con-sequently, such food packaging should not contain OBAs, and food packagingmade from recycled fibers should be tested to ensure that there is no migra-tion of OBAs into food.[3] Because the compounds are soluble in water, theycan accumulate in natural ground and surface waters and they are not easilydegradable by environmental biological systems.[4] Hence, their analysis anddetermination in various matrices is important.

OBAs were originally analyzed by thin-layer chromatography (TLC) andspectrophotometric techniques.[5,6] However, these techniques are affected byvarious interfering species and suffer from low selectivity. Gas chromatography(GC) has limited applications in the analysis of the OBAs used in the pulpand paper industry because of their high molecular weights and low volatilityinduced by the presence of hydroxyl, amino, and disulfonate groups, evenafter extensive derivatization. Capillary zone electrophoresis (CZE) with UVand fluorescence detectors has also been used in the analysis of aromaticsulfonates[7] and for the determination of FWAs in paper and board packagingafter using hot water extraction.[8] We have previously used spectrofluorometryto measure the migration of OBAs from food packaging made from recycledfibers.[9] The method works well for monitoring of total OBAs but cannotidentify the chemistries of the present OBAs.

Liquid chromatography–mass spectrometry (LC-MS) has been used forbetter characterization of the chemistries of OBAs. Ogura et al.[10] devel-oped a technique for characterization of optical brighteners from five groupsof stilbene, biphenyl stilbene, pyrazoline, oxazole, and coumarin deriva-tives using LC-MS. Nageswara Rao et al.[11] analyzed OBA compoundssuch as 4,4′-dinitrostilbene-2,2′-disulfonic acid, 4-amino-4′-nitrostilbene-2,2′-disulfonic acid, and 4,4′-diaminostilbene-2,2′-disulfonic acid and studied theirdegradation products using electrospray ionization–mass spectrometry (ESI-MS). They also studied[12] separation and determination of aromatic sulfonatesin an aquatic environment using ESI-MS. In another study,[13] OBAs were in-vestigated qualitatively and quantitatively by solid-phase extraction followingby high-performance liquid chromatography (HPLC)-ESI-MS (ion trap) ap-plying di-n-hexylammonium acetate as the ion-pairing reagent in the mobilephase. Chen and Ding[14] used hot water and solid-phase extraction followedby mass spectrometric analysis to measure OBAs in paper and clothes. Allof the above ESI-MS detections used negative-ion modes. Stoll and Giger[15]

developed a method for the extraction and quantitative determination of threedetergent-derived OBA isomers in lake sediments and surface waters by HPLCand fluorescence detection after postcolumn UV irradiation.

LC-QTOF-MS, which is a hybrid of LC interfaced to a mass spectrometercontaining two mass analyzers in parallel, a quadrupole (Q), and time of flight(TOF) analyzer, provides good mass accuracy and sensitivity for determinations

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Analysis of Optical Brightening Agents 45

of low- and high-molecular-weight compounds. The instrument is also capableof MS-MS analysis in which high-molecular-weight compounds can be se-lected as their precursor ions by the quadrupole mass analyzer and fragmentedin a collision cell to produce and identify its fragment ions in a time-of-flightmass analyzer. This technique has been applied to the analysis of a wide range offunctionally complex compounds including phosphorylated peptides,[16] pro-tein tyrosine-O-sulfated peptides,[17] alkaloids,[18] and flavonoids,[19] but it hasnot yet been applied in the investigation of OBAs. In the present study we de-scribe for the first time the separation and accurate mass determination of fourstilbene derivative OBAs including 4,4′-diamino-2,2′-stilbene-disulfonic acid(DADS), DOBA, TOBA, and HOBA in the molecular range of 370–1369 Da inboth negative- and positive-ion modes by LC-ESI-QTOF-MS. The extractionand analysis of OBAs present in different commercial copy-grade papers con-taining OBAs and the observation of isomerization occurring in some structuresunder natural light were also undertaken.

EXPERIMENTAL

Chemicals

Ammonium acetate (98%), DADS (85%), and fluorescent brightener 28(DOBA) were purchased from Sigma Chemical Co. (St. Louis, MO). Tetra-sulfonated OBA (TOBA), which was a commercial formulation, KalBitec,was kindly supplied by Kalamazoo Chemicals (Richland, MI), and hexasul-fonated OBA (Leucophor BCS), or HOBA, was kindly supplied by ClariantInc. (Charlotte, NC; material number: 15363624156). LC-MS-grade acetoni-trile, ethanol, and water were obtained from Honeywell Burdick & Jackson(Muskegon, MI). The concentrations of the standards were 8 mg/10 g of ace-tonitrile/water (35%/65%). However, because of the low solubility of DADSand the possibility of clogging the LC syringe, the standard solutions were fil-tered on polytetrafluoroethylene (PTFE) filters (0.20 µm pore size); therefore,the concentration of DADS was lower than expected.

OBA Extraction from Paper Samples

Hot water extraction of OBAs from paper products was initially chosen be-cause other studies[8,14] have shown satisfactory spike recoveries of OBAsfrom paper. The present study goes further by examining aqueous alcoholicand aqueous acetonitrile as extraction solvents as well. Paper samples were cutinto small pieces and known weights were extracted using a SoxtecTM 2050extraction apparatus (FOSS North America, Inc., Eden Prairie, MN). Operation

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46 F. Shadkami, R. Helleur, and B. B. Sithole

and use of a Soxtec apparatus has been described previously.[20] Several sol-vents, namely, ethanol/water (50/50%), methanol/water (50/50%), acetoni-trile/water (50/50%), water, and ethanol, were evaluated to ascertain the bestextraction efficiency for characterizing OBA present in real samples. The finalpaper extracts were analyzed by LC-QTOF-MS. Three different paper sampleswith differing brightness levels were analyzed for OBAs including copy-gradeprinting paper with a brightness of 92, a card paper with a brightness of 86, anda darker paper with a brightness of 79.

LC-QTOF-MS

An accurate-mass LC-QTOF LC/MS (Model 6520, Agilent Technologies, Mis-sissauga, ON, Canada) was used for analysis. The OBAs were separated ona C18 HPLC column (Zorbax Eclipse plus, 2.1 × 50 mm, 1.8 µm, AgilentTechnologies, Mississauga, ON, Canada). The chromatographic system wasan Agilent liquid chromatograph, 1200 Series, using a gradient elution withsolution A (water, 5 mM ammonium acetate, pH = 7.2) and solution B (50%acetonitrile:50% methanol) at a flow rate of 0.2 mL/min. The gradient programused was as follows: 10% B increased to 60% B in 16 min, then 90% B in 25 min,and finally 95% B in 27 min. The ion source was an ESI. The source and otherMS parameters were as follows: gas temperature = 350◦C; drying gas flow =10 L/min; nebulizer gas pressure = 40 psig; Vcap = 4000 V; MS TOF frag-mentor = 200 V; skimmer voltage kept at 65 V. The mass spectrometer wasoperated in both positive and negative modes. MS data were acquired in bothcentroid and profile modes and processed using Agilent MassHunter software.In MS/MS studies, a fixed collision energy of 60 V was applied and the refer-ence masses (112.0509 and 922.0092) were excluded.

Exposure of OBA Standards to Light Irradiation

All four OBA standards (DADS, DOBA, TOBA, and HOBA) in concentrationsof 10, 100, and 1000 ppm in water were exposed to light for one month. Thesource of the light was normal sunlight exposure during the day and cool whitefluorescence lamp at night. The goal was to examine the chemical stability ofOBA standards in the light and the occurrence of isomerization.

RESULTS AND DISCUSSION

Mass Spectral Characterization of OBAs

As shown in Figure 1, two of the OBA standards were sodium salts of sulfonicacid (TOBA and HOBA) and the other two were sulfonic acids (DADS and

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Analysis of Optical Brightening Agents 47

Figure 2. LC-QTOF-MS: (a) total ion chromatogram for mixture of four optical bright-ening standards including DOBA-U (an unknown impurity along with DOBA) and(b) extracted ion (+ mode) chromatogram for HOBA.

DOBA). Under the chromatographic conditions employed, it is expected thatall of the structural forms would be in their full acidic form. The MS datasupport this. The chromatographic conditions (mobile phases, gradient elution,and column type) were optimized to achieve adequate resolution of the OBAcompounds as illustrated in Figure 2a. However, coelutions of DADS andHOBA could not be resolved. Figure 2b demonstrates that the extracted ionchromatogram for an m/z value of 1237.1060 isolates the HOBA peak. Thischromatogram is the result of LC separation and ESI-QTOF mass detection inthe positive mode, which is in contrast to other studies in which only the negativemode was used.[10–12] The different retention times of the OBA standards canbe explained by the differences in the molecular weights of the compounds,the interaction of acidic sites of the OBA with the C18 nonpolar column, andthe interaction of polar hydroxyl and amino groups with the column and theirdifferent behaviors in multicomponent gradient elution.

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48 F. Shadkami, R. Helleur, and B. B. Sithole

Figure 3. QTOF mass spectra of DADS in (a) positive-ion and (b) negative-ion mode.

The anticipated mass spectrum of DADS was observed in both positive andnegative modes as shown in Figure 3. The mass-to-charge ratios correspondingto [M + H]+, 371.0376, and [M − H]−, 369.0211, were observed. The molarmass of the chemical, based on its empirical formula (C14H14N2O6S2), is370.0293. The mass accuracy of observed vs. calculated data obtained with theQTOF-MS for all observed ions are given in Table 1.

Two chromatographic peaks for the standard DOBA were observed: DOBAand DOBA-U (DOBA-unknown; Figure 2a). The molar mass of DOBA, withthe empirical formula of C40H44N12O10S2, is 916.2745. Analysis of the DOBApeak (actual DOBA) in the positive-ion ESI mode showed m/z adducts of917.2845 and 459.1456, which represent a singly charged ion of [M + H]+ and

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Tabl

e1.

Mas

ssp

ectr

ada

taan

das

sign

men

ts

Ret

entio

ntim

eM

olec

ular

m/z

m/z

Err

orC

ompo

und

(min

)w

eigh

t(O

bser

ved)

(Cal

cula

ted)

(ppm

)A

ssig

nmen

tPo

lari

ty

DA

DS

1.01

370.

0293

371.

0376

371.

0372

1.1

[M+

H]+

(+)

369.

0211

369.

0215

−1.1

[M−

H]−

(−)

DO

BA

16.5

091

6.27

4591

7.28

4591

7.28

182.

9[M

+H

]+(+

)45

9.14

5645

9.14

511.

1[M

+2H

]2+(+

)91

5.26

5491

5.26

61−0

.76

[M−

H]−

(−)

457.

1305

457.

1299

1.3

[M−

2H]2−

(−)

DO

BA

-U14

.30

928.

2901

929.

3031

(+)

465.

1562

(+)

927.

2911

(−)

463.

1423

(−)

TO

BA

9.89

1076

.188

110

77.1

941

1077

.195

9−1

.7[M

+H

]+(+

)53

9.10

1653

9.10

19−0

.56

[M+

2H]2+

(+)

1075

.179

710

75.1

803

−0.5

6[M

−H

]−(−

)53

7.08

7753

7.08

61−2

.9[M

−2H

]2−(−

)Pr

ecur

sor

ion

MS/

MS

1043

.171

010

43.1

742

−3.1

[M−

2OH

+H

](+

)M

S/M

Sof

1077

.194

1of

TO

BA

963.

1002

963.

1040

−3.9

[104

3.17

10−

SO3]

(+)

MS/

MS

HO

BA

1.01

1236

.101

712

37.1

060

1237

.109

6−2

.9[M

+H

]+(+

)61

9.05

8061

9.05

87−1

.1[M

+H

]2+(+

)

49

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50 F. Shadkami, R. Helleur, and B. B. Sithole

Figure 4. QTOF mass spectra of DOBA: (a) positive-ion and (b) negative-ion mode.

a doubly charged ion of [M + 2H]2+ (Figure 4a), respectively. In the negative-ion mode DOBA yielded two ions with m/z of 915.2654 and 457.1305 thatcorrespond to [M − H]− and [M − 2H]2− (Figure 4b), respectively. On theother hand, for DOBA-U, negative-ion ESI yielded a simple spectrum with ionsof m/z 927.2911 and 463.1423 (Figure 5a). A similar pattern was observed inthe positive-ion mode but with the addition, instead of subtraction, of a proton(Figure 5b). This finding suggests that the molecular weight of DOBA-U canbe 928.2901. This structure is unknown, but because of its molecular weightand chromatographic behavior it is considered an impurity in the industrialproduction of DOBA; however, the molecular weight could correspond to[M − 2OH + 2Na].

The chromatographic results for TOBA with a molar mass of 1076.1881(C40H44N12O16S4) is shown in Figure 2a. The standard TOBA sodiated saltconverts to its acidic form when injected into the LC mobile phase. The mass

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Analysis of Optical Brightening Agents 51

Figure 5. QTOF mass spectra of DOBA-U (unknown DOBA standard) in (a) negative-ion and (b) positive-ion mode.

spectrum of the TOBA peak at the retention time of 9.89 min gives m/z =1075.1797 in the negative-ion mode and m/z = 1077.1941 in the positive-ionmode. In the negative-ion mode m/z = 1075.1797 could correspond to [M −H]−, whereas m/z = 537.0877 would correspond to a doubly charged ion of[M − 2H]2− (Figure 6b). Also, in the positive-ion mode the m/z = 1077.1941could correspond to [M + H]+ and m/z = 539.1016 would correspond to [M +2H]2+ (Figure 6a). Mass-to-charge values of 1075.1797 in the negative-ionmode and 1077.1941 in the positive-ion mode are strong markers of TOBA.

The molar mass of HOBA, based on the empirical formula ofC40H44N12O22S6, is 1236.1017. The HOBA assignment in the positive-ionmode gave m/z values of 1237.1060 and 619.0580 (Figure 6c). These m/zvalues would correspond to [M + H]+ and [M + H]2+.

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52 F. Shadkami, R. Helleur, and B. B. Sithole

Figure 6. QTOF mass spectra of TOBA in (a) positive-ion and (b) negative-ion modeand (c) HOBA in positive-ion mode.

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Analysis of Optical Brightening Agents 53

In Table 1 all calculated masses were reported using QTOF software masscalculator, and the ppm mass error was calculated based on [(Observed mass− Calculated mass)/Calculated mass] × 106.

MS-MS of OBA Standards

A representative precursor ion MS-MS mass spectrum in this study was TOBAin the positive-ion mode. The precursor ion at 1077.1941 was selected andsubjected to low-energy collision showing two major ions at m/z = 1043.1710and m/z = 963.1002. These ions are believed to correspond to [M − 2OH +H] and [1043.1710 − SO3], respectively (Figure 7). Thus, the structural infor-mation that can be obtained by MS-MS data of these OBA compounds is dueto the presence of hydroxyl and sulfone groups.

Figure 7. (a) QTOF mass spectrum of TOBA in positive-ion mode and (b) precursorion MS/MS of m/z 1077.1941 of TOBA.

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Exposure of OBA Standards to Light

All four OBA standards (DADS, DOBA, TOBA, and HOBA) were exposedto light for one month. The standards were analyzed by LC-QTOF-MS afterexposure. DADS and HOBA did not show any isomerization; however, thechromatogram of DOBA showed an isomer at retention time of 16.0 min.TOBA also showed an isomer (TOBA′) at retention time of 5.3 min in achromatogram similar to that shown in Figure 8. The mass spectra of trans andcis isomers were the same. Only the peak area for the main TOBA and DOBApeaks were decreased by about 30% after exposure to light. This is importantfor papermaking because the cis and trans forms of OBAs affect the brightnessof the papers.[17] Also, this is important for quantitative measurements of OBAswherein the presence of both cis and trans forms of the OBAs may not be takeninto account. This will lead to erroneous calculations if the quantitation is donebased on only one form of the OBAs.

Extraction of OBAs from Paper Samples

The extraction efficiency was calculated based on the sum of the peak areas forboth TOBA and its TOBA′ isomers (Figure 8) relative to the peak area of a ref-erence TOBA standard (TOBA). Afterwards, the weight percentage of TOBAand TOBA′ in a known weight of paper sample was calculated after consider-ing the concentration factor. All extraction solvents except ethanol were ableto qualitatively extract the OBAs present in the paper. The only OBAs found inthe paper products under study were TOBA or TOBA′. The amounts extracted

Figure 8. LC-QTOF-MS total ion chromatogram for a printing paper extract (TOBAand TOBA′ are two isomers).

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Analysis of Optical Brightening Agents 55

based on sample weight were low; the highest was 0.048% using hot water.Our study did not quantitatively measure the remaining OBA on the paper.This could be accomplished in future studies using a fluorescence spectrometercapable of reflectance to quantify the amount of remaining fluorescent OBAs.

The percentage extraction for the various solvents were water (0.048 ±0.002%) > methanol/water (50%) (0.030 ± 0.004%) > acetonitrile/water(50%) (0.025 ± 0.003%) > ethanol/water (50%) (0.015 ± 0.003%). The valuesin parentheses indicate the weight percentage of TOBA and TOBA′ in printingpaper (brightness 92) for seven replicates. The relative polarity of the OBAs,based on their different functional groups, supports the descending order ofextraction efficiency from lower organic content (i.e., water) to higher organiccontent (i.e., ethanol); however, it should be noted that other researchers[21]

have mentioned the use of dimethylformamide (DMF) to extract OBAs frompaper samples.

Further studies should be undertaken on paper products where aluminumsulfate and other additives are used as cationic fixatives for OBAs onto fibersand, therefore, OBAs maybe more difficult to extract. The employment of acidicextraction conditions could thus be a good solution to overcome the chemicalfixation and to assist in weakening the hydrogen bonds between OBAs andcellulose.

Analysis of OBAs in high-brightness paper (brightness 92) showed thatthe paper contained two stereochemical isomers of the acidic form of tetrasul-fonated OBA (TOBA and TOBA′) as illustrated in Figure 8. Seven replicatedanalyses indicated that the paper contained 0.048 ± 0.002% OBA (w/w%). Twolower brightness papers, B-86 and B-79, contained the same OBA at 0.008 ±0.001% and 0.002 ± 0.001%. Surprisingly, a significant amount of DOBA(0.053 w/w%) was found in the paper with the lowest brightness (B-79). Thisis because the paper was made from mechanical pulp, unlike the other papersthat were made from fully bleached kraft pulps. The result illustrates that ad-dition of OBAs to mechanical pulps is less efficient in induction of brightnessthan addition of similar dosages of OBAs to bleached kraft pulp.[22,23]

Analysis of papers exposed to light for a month in a simulated, intense coolwhite fluorescence chamber showed no further isomerization of the OBAs.

CONCLUSIONS

Optical brightening agents were separated and analyzed by LC-QTOF-MS.For all OBAs (DADS, DOBA, TOBA, and HOBA) the molecular ions cor-responded mainly to [M + H]+ and [M − H]− in positive and negative-ionESI, respectively. This work advances the applications of OBAs in industryin which OBAs can be qualitatively and quantitatively analyzed. Analysis ofcommercial copier-grade papers showed the presence of two stereochemicalisomers of tetrasulfonated OBA or TOBA, which is the most economical OBA

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56 F. Shadkami, R. Helleur, and B. B. Sithole

additive in the pulp and paper industry. It was found that, for the paper productsused in this study, the descending order of extraction efficiency was from lowerorganic content (i.e., water) to higher organic content (i.e., ethanol). However,in future work it may be prudent to explore acidic extraction conditions, orother organic solvents such as DMF, to ascertain their extraction efficiency.Further research is required on extraction techniques and on a wider range ofpaper products before routine extraction and analysis of OBAs is established.This should include a method that can semiquantitatively measure the amountof OBA remaining on the paper after extraction by measuring fluorescence; forexample, a fluorescence reflectance spectrometer.

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