CHAPTER 22 • Organic Matter Sources and Dynamics in Adriatic Coastal Waters
473
recovery (R, as a percentage of the initial DOC) was calculated according to Dai et al.
(1998). For the 1 kDa cartridge, R % ranged from 87 to 120% (with an average of
101 ±8%), while for the 10 kDa cartridge, R % ranged from 83 to 117% (with an average
of 100 ±8%). Overall recovery in our ultrafiltration system was close to 100% (±1O%)
and was within the ranges reported in the literature (Guo et al.1994; Benner et al.1997;
Dai et al. 1998), indicating that contamination or loss of organic carbon were minimal. While this fractionation procedure is probably the best one available at the moment
and the most widely used, we must note that interactions at membrane surface may lead
to conformation changes, irreversible adsorption and lor self-coagulation of small molecules resulting in an overestimation of COC (Buftle and Leppard 1995; Guo et al. 2000).
Dissolved Free (DFAA) and Total Dissolved (TDAA) Amino Acids. Samples for total
dissolved amino acids (TDAA) were analyzed after liquid hydrolysis in 6 N HCl at 110°C
for 22 h (modified from Parson et al. 1984). 2 ml samples were placed in glass ampoules
(acid washed and muffled for 3 hours at 500°C), sealed under a flow of N2• Each vial
was spiked with a-aminoadipic acid, norvaline and hydroxylysine as recovery standards of the acidic, neutral and basic classes, respectively (Cowie and Hedges 1992), and
2 ml of ultrapure 12 N HCl. Hydrolysates were vacuum dried and then redissolved in
milli-Q water to achieve a neutral pH. DFAA were analyzed without preliminary hydrolysis of the samples.
DFAA and TDAA were determined by high performance liquid chromatography
(HPLC-Perkin Elmer Series IV) and fluorimetric detection (Perkin Elmer LS4) with a
gradient system of methanolltetrahydrofuran (90:10 v/v) acetate buffer, following
derivatization with o-phthaldialdehyde (OPA) and 2-mercaptoethanol (Lindroth and
Mopper 1979, as modified in Pettine et al.I999); 21 individual amino acids were separated according to this procedure (see Fig. 22.1). Daily standards in aged sea water,
samples and blanks (aged sea water) were run in triplicate. The average TDAA blank
amounted to 62 ±40 nM and was mainly due to glycine, aspartic acid, serine and alanine. Recovery for most of the amino acids was within ±1O% except for asparagine,
glutamine, tryptophane, histidine and methionine. In the analysis of free amino acids, glutamic acid, glutamine, aspartic acid and asparagine are separated into four distinct peaks, but the acid hydrolysis performed for analyzing total dissolved amino acids
causes the deamination of asparagine (asn) and glutamine (gIn) to form aspartic acid
(asp) and glutamic acid (glu) plus ammonium. Therefore, in the TDAA analysis, the
amino acids pairs (asn + asp and gIn + glu) give only two peaks instead of four. Tryptophane is unstable during acid hydrolysis and may not be detected. Histidine is also
partially modified by acid hydrolysis and the recovery is only 20-30% (Keil and
Kirchman 1991). DFAA and TDAA are expressed as individual amino acids (nM) and
as the sum of the amino acidic molar carbon (DFAA-C or TDAA-C) and nitrogen
(DFAA-N or TDAA-N) units.
Total Dissolved Carbohydrates. Total dissolved carbohydrates (TDCHO), including
mono-, oligo- and polysaccharides, were analyzed by the MBTH method after a preliminary hydrolysis with 0.09 N HCl (20 h at 100°C) (Burney and Sieburth 1977). This
hydrolysis procedure was preferred to the stronger H2S04 attack more recently proposed
(Pakulski and Benner 1992; Mopper et al. 1992), mainly because it has been extensively
used in estuarine and coastal waters, thus making the comparison with previous find-
473
recovery (R, as a percentage of the initial DOC) was calculated according to Dai et al.
(1998). For the 1 kDa cartridge, R % ranged from 87 to 120% (with an average of
101 ±8%), while for the 10 kDa cartridge, R % ranged from 83 to 117% (with an average
of 100 ±8%). Overall recovery in our ultrafiltration system was close to 100% (±1O%)
and was within the ranges reported in the literature (Guo et al.1994; Benner et al.1997;
Dai et al. 1998), indicating that contamination or loss of organic carbon were minimal. While this fractionation procedure is probably the best one available at the moment
and the most widely used, we must note that interactions at membrane surface may lead
to conformation changes, irreversible adsorption and lor self-coagulation of small molecules resulting in an overestimation of COC (Buftle and Leppard 1995; Guo et al. 2000).
Dissolved Free (DFAA) and Total Dissolved (TDAA) Amino Acids. Samples for total
dissolved amino acids (TDAA) were analyzed after liquid hydrolysis in 6 N HCl at 110°C
for 22 h (modified from Parson et al. 1984). 2 ml samples were placed in glass ampoules
(acid washed and muffled for 3 hours at 500°C), sealed under a flow of N2• Each vial
was spiked with a-aminoadipic acid, norvaline and hydroxylysine as recovery standards of the acidic, neutral and basic classes, respectively (Cowie and Hedges 1992), and
2 ml of ultrapure 12 N HCl. Hydrolysates were vacuum dried and then redissolved in
milli-Q water to achieve a neutral pH. DFAA were analyzed without preliminary hydrolysis of the samples.
DFAA and TDAA were determined by high performance liquid chromatography
(HPLC-Perkin Elmer Series IV) and fluorimetric detection (Perkin Elmer LS4) with a
gradient system of methanolltetrahydrofuran (90:10 v/v) acetate buffer, following
derivatization with o-phthaldialdehyde (OPA) and 2-mercaptoethanol (Lindroth and
Mopper 1979, as modified in Pettine et al.I999); 21 individual amino acids were separated according to this procedure (see Fig. 22.1). Daily standards in aged sea water,
samples and blanks (aged sea water) were run in triplicate. The average TDAA blank
amounted to 62 ±40 nM and was mainly due to glycine, aspartic acid, serine and alanine. Recovery for most of the amino acids was within ±1O% except for asparagine,
glutamine, tryptophane, histidine and methionine. In the analysis of free amino acids, glutamic acid, glutamine, aspartic acid and asparagine are separated into four distinct peaks, but the acid hydrolysis performed for analyzing total dissolved amino acids
causes the deamination of asparagine (asn) and glutamine (gIn) to form aspartic acid
(asp) and glutamic acid (glu) plus ammonium. Therefore, in the TDAA analysis, the
amino acids pairs (asn + asp and gIn + glu) give only two peaks instead of four. Tryptophane is unstable during acid hydrolysis and may not be detected. Histidine is also
partially modified by acid hydrolysis and the recovery is only 20-30% (Keil and
Kirchman 1991). DFAA and TDAA are expressed as individual amino acids (nM) and
as the sum of the amino acidic molar carbon (DFAA-C or TDAA-C) and nitrogen
(DFAA-N or TDAA-N) units.
Total Dissolved Carbohydrates. Total dissolved carbohydrates (TDCHO), including
mono-, oligo- and polysaccharides, were analyzed by the MBTH method after a preliminary hydrolysis with 0.09 N HCl (20 h at 100°C) (Burney and Sieburth 1977). This
hydrolysis procedure was preferred to the stronger H2S04 attack more recently proposed
(Pakulski and Benner 1992; Mopper et al. 1992), mainly because it has been extensively
used in estuarine and coastal waters, thus making the comparison with previous find-