Leo Wang
Thermo Fisher Scientific, Sunnyvale, CA, USA
Low Molecular Mass Organic Acids (LMMOAs) are
degradation products during pretreatment processes for
biofuel production that are known to inhibit microbial
fermentation processes, and thus reduce the conversion
efficiency of biomass to biofuels. To better understand and
optimize the conversion process, an IC/MS method to
quantitate LMMOA profiles during biofuel production
was developed. The IC/MS method is particularly useful
in separating and profiling close eluters using the power of
high performance anion exchange chromatography
coupled with multiple SIM scans to identify over
30 compounds in a single analytical run. LMMOAs
were detected and reported with various concentrations
ranging from 0.140 ppm to > 200 ppm.
Conditions
System:
Thermo Scientific™ Dionex™ ICS-2000
Columns:
Thermo Scientific™ Dionex™ IonPac™ AS11-HC
analytical column with AG11-HC guard, 2 mm
Eluent:
Hydroxide gradient:
Time/minConcentration/mM
–10 1.0
01.0
81.0
1815
2830
3860
40 1.0
Eluent Source: EGC II KOH
Flow Rate:
0.38 mL/min
Inj. Volume:
25 µL (or mL, etc.)
Detection:
Suppressed Conductivity
Mass Spectrometric Detection
For Suppressed Conductivity Detection:
Suppressor: Thermo Scientific™ Dionex™ ASRS™ 300
Anion Self-Regenerating Suppressor™, 2 mm
Regenerant: Deionized water
Regenerant Flow Rate: 0.5 mL/min
Mass Spectrometric Detection:
Instrument: Thermo Scientific™ MSQ Plus™
Ionization Method: Electrospray Ionization (ESI) source
Scan Mode: Negative Selected Ion Monitoring (SIM)
Nebulizer Gas: Nitrogen at 80 psi
Probe Temperature: 450 °C
Needle Voltage: 3000 V
Appli cat i on Br i e f 1 0 4
Determination of 32 Low Molecular
Mass Organic Acids in Biomass by
Ion Chromatography Mass Spectrometry
2
propionate
glycolate
3 pyruvate
Analyte
4
butyrate
lactate 5
isovalerate valerate
oxalate
6
malonate
2-methyllactate
7
IStd-valerate-d9
maleate
8
methylmalonate
9
10
succinate 11
2-hydroxyvalerate
13
fumarate
12
glutarate
malate
14
IStd-glutarate-d6
15
16 a-ketoglutarate
adipate
tartrate
cis-aconitate
17
transaconitate
18
19
isocitrate
citrate
quinate
24
SIM
m/z
191.1
195.1
89.0
103.1
59.0
75.0
73.0
45.0
87.0
193.0
117.1
87.0
101.1
110.0
101.1
193.0
193.0
137.0
131.0
209.0
145.1
117.0
133.0
117.0
103.0
149.0
115.0
145.1
89.0
115.0
131.0
191.0
195.0
191.0
173.0
173.0
20
21
galacturonate
2-keto-D-gluconate
5-keto-D-gluconate
gluconate
mucate
quinate
gluconate
lactate
2-methyllactate
acetate
glycolate
propionate
formate
butyrate
2-keto-D-gluconate
2-hydroxyvalerate
pyruvate
isovalerate
IStd valerate-d9
valerate
galacturonate
5-keto-D-gluconate
IStd glutarate-d6
glutarate
mucate
adipate
succinate
malate
methylmalonate
malonate
tartarate
maleate
a-ketoglutarate
oxalate
fumarate
oxalacetate
citrate
IStd citrate-d4
isocitrate
cis-aconitate
trans-aconitate
R.T.
min
6.9
7.7
8.2
8.2
8.8
8.8
10.2
11.5
12.2
12.2
12.5
12.9
13.1
14.0
14.3
14.4
19.1
21.5
21.6
21.6
21.7
21.9
22.1
22.4
22.8
22.8
23.7
24.6
25.3
25.6
28.5
33.5
33.5
34.3
35.1
36.7
IStd citrate-d4
22
23
25
0
5
10
15
25
30
35
40
0
5
10
15
20
25
Retention Time (min)
30
35
40
20
18.0
µS
–2.0
25609
Figure 1. CD and MS SIM chromatograms of 32 small organic acids.
1. Each SIM chromatogram is normalized to the greatest peak in that channel.
2. 100 ppb for each internal standard.
3. MS detection: 50 ppb for each analyte except for cis- and trans-aconitate whose combined total concentration is 50 ppb
Conductivity detection: 5 ppm for each analyte, aconitates have a total concentration of 5 ppm.
4. SIM channels are sorted by increasing m/z from top to bottom.
5. SIM scan numbers 1 and 2 (formate and acetate) are not shown due to weak response; scans 4–23 are identified and summarized in the table above.
Conclusion
Appli cat i on Br i e f 1 0 4
IC/MS is a robust and highly selective method for
monitoring LMMOAs and other trace-level fermentation
inhibitors present in complex matrices, such as fermentation broths. The power of IC/MS detection enables
quantitation of more than 30 closely eluting compounds
in real-world samples which are difficult to fully resolve
chromatographically.
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