CN108562670B - A kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method - Google Patents
A kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method Download PDFInfo
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Abstract
A kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method, chromatographic condition are that quantitative detection wavelength is 223 nm, and chromatographic column is Agilent SB-C18, and column oven temperature is 35 DEG C；Using acetonitrile and water as mobile phase, the fixed flow rate of 0.8 mL/min；Gradient elution program are as follows: EP (end of program) when being reduced to 10%, 34 minutes again when rising to 70%, 32 minutes when acetonitrile volume ratio is 10%, 30 minutes at 0 minute.With qualitative, quantitative, accurate, remolding sensitivity direct method improves at least two order of magnitude, and can measure butyric acid several monocarboxylic acid derivative-liquid phase chromatography detection methods proposed by the present invention, and advantage is clearly.
Description
Technical field
The invention belongs to a kind of organic acid detection techniques of the related fieldss such as agricultural, ecology and environment, more particularly to ecology
The analysis of highly sensitive instrument and detection of several low content monocarboxylic acids in ambient water sample, plant rhizosphere sample.
Background technique
Organic acid is widely present in nature, rhizosphere area small molecular organic acid mostly come from plant and animal residues decomposition,
The conversion of microbial metabolism, the secretion of root system of plant and decomposition and the organic matter being manured into soil.It is generally existing in soil
A kind of functionality organic matter is that carbon in soil most enlivens form, can pass through the influence of the complex mechanisms such as chelating and ligand exchange
The chemistry and biology process of soil, to the formation differentiation of soil, the validity of plant rhizosphere mineral nutrient, acid soil aluminium poison
Alleviate and a variety of soil courses such as heavy metal activity generate profound impact, and influences a variety of physiological and ecological process of plant strongly,
It adjusts adaptation of the plant to environment and the resistance to poor environment, its generation is directly connected with soil microbe quantity and activity
System, and it is closely related with soil types, root ring border etc..Currently, the small molecular organic acid master detected in plant rhizosphere sample
It to include 2 major class of aliphatic and aromatic series, aromatic organic acid (also commonly referred to as allelochemical) mainly includes having aroma
Taste: the phenolic acid compounds such as benzoic acid, salicylic acid, P-hydroxybenzoic acid, vanillic acid, coumaric acid；Aliphatic organic acid is then
Specifically include that formic acid, acetic acid, lactic acid, malic acid, succinic acid, tartaric acid, citric acid, oxalic acid etc..Wherein, acetic acid is in ecosystem
Intermediate compound is important during the carbon nitrogen cycle of system, has certain pass with GHG carbon dioxide and methane
Connection, therefore, the monitoring to acetic acid are also the emphasis of correlative study person's concern.
Although most of organic acid has positive effect to the growth of plant in rhizosphere, simultaneously it is also noted that some organic acids
Excess and several aliphatic monocarboxylic acids in bring adverse effect, such as plant rhizosphere --- formic acid, lactic acid, acetic acid, propionic acid,
Butyric acid etc., amount appropriate has certain positive influences, but plant rotten etc. is likely to result in after content increases to certain value,
It in turn results in and hinders the adverse effects such as plant growth；In addition, the content of these types of organic acid is usually lower, instrument detection sensitivity
Difference, measurement difficulty is larger, therefore must have the Accurate Determining method of such suitable acid, for Related Research Domains such as agricultural or ecologies
Scientific research, scientific worker provide reliable reference.
Aliphatic organic acid analysis method has traditional colorimetric method, spectrophotometry, enzyme process etc., but these methods are due to behaviour
Make step complexity, the disadvantages such as the rate of recovery is low limit its extensive use.Red, orange, green, blue, yellow (ROGBY) is one kind with fastest developing speed in recent years point
From analysis means, and the most effectual way of separation and detection organic acid.Currently, the detection of organic acid mainly uses gas-chromatography
Method (GC/FID) or gas chromatography mass spectrometry (GC/MS), high performance liquid chromatography (HPLC/UV), the chromatography of ions (IC-UV/
ELCD) improved well with capillary electrophoresis (CE/UV), the sensitivity of detection compared to conventional method.
GC/FID method or GC/MS method measure organic acid, and separating effect is generally fine on a column for sample, but to sample
The complex time-consuming of pre-treatment, sample need to examine by complicated derivatization treatment (usually esterification or silanization etc.)
It surveys, this can cause the loss of object to a certain extent and reduce the rate of recovery, and it is precision, more less reproducible, simultaneously because
The introducing of derivatization reagent, chromatogram also can be more complicated.
HPLC method and IC method sample pre-treatments are typically more simple, after high-content organic acid sample even can be filtered directly
Sample introduction, while the separating effect of organic acid on a column is preferable, this makes both methods in organic acid analysis using non-
Often extensively.But their major defect is the accurate qualitative and quantitative inspection that complicated ingredient in actual samples will affect object
It surveys, low content sample is then difficult to measure.If chromatographic column is expensive, and right using dedicated organic acid measuring column
The complex pretreatment of sample, need to be by sample except decationizing etc., and equally exists the problem of instrument detection sensitivity difference；It is using
When IC method analyzes organic acid, some organic acids are influenced by water negative peak, it is difficult to carry out qualitative and quantitative analysis.The main table of CE rule
Now seldom for sample dosage, analysis time is shorter, and interference is small compared with HPLC method and IC method, but the stability ratio HPLC method and IC of instrument
Method is more poor, the problem low there is also detection sensitivity for low content organic acid sample, and operating cost is also relatively high.
Summary of the invention
The technical issues of solution: the present invention is for several low content monocarboxylic acid (formic acid, acetic acid, third in above-mentioned rhizosphere sample
Acid and butyric acid) determination techniques problem, a kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method is provided.
Technical solution: a kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method, step are as follows: chromatographic condition: quantitative detection
Wavelength is 223nm, and chromatographic column is Agilent SB-C18 (250mm × 4.6mm, 5 μm), and column oven temperature is 35 DEG C；With acetonitrile
With water as mobile phase, the fixed flow rate of 0.8mL/min；Gradient elution program are as follows: acetonitrile volume ratio is 10% at 0 minute,
EP (end of program) when being reduced to 10%, 34 minutes when rising to 70%, 32 minutes at 30 minutes again；Reagent solution needed for derivative reaction
Preparation: EDC solution 30-60mmol/L；MES solution 30-60mmol/L is 5-6.7 with NaOH solution tune pH value；NHS solution
20-90mmol/L；Derivative reagent amine compound solution 30-60mmol/L, with acetone or acetonitrile hydrotropy；Standard solution is prepared:
Prepare 4 kinds of monocarboxylic acid hybrid standard mother liquors are as follows: formic acid, acetic acid, propionic acid, butyric acid are 100mg/L；Derivative reaction: standard is molten
The derivative reaction of liquid: it takes 4 kinds of monocarboxylic acid hybrid standard mother liquors of 0-1mL to be added in the scale test tube of 5mL respectively, then adds respectively
Enter the MES solution of 0.1-0.6mL EDC solution, 0.2-1.0mL, 0.1-0.5 mL NHS solution is added after twenty minutes, reacts molten
The pH value of liquid controls between 5.0-6.0, after standing reaction 0.5-1 hours, adds the derivative reagent amido of 0.1-1.0 mL
Compound solution stands reaction solution 10-20 hours, is finally settled to 5mL with ultrapure water, instrument is to be measured；Entire reaction process
Complete at room temperature, carboxylic acid in reaction system: EDC:NHS molar ratio=1:(1~4): the range of (1~2), carboxyl: amine
The molar ratio of substratess matter is between 2-4；The derivative reaction of sample solution: derivative reaction of the step with standard solution, sampling
Measure 0.5-1.0mL；Assay: precision draws the standard solution and each 20 μ L of sample solution after derivative reaction, injects liquid phase
Chromatograph measures to get result.
The optimization concentration of each derivatization reagent are as follows: EDC solution 45mmol/L 0.5mL；MES solution 60mmol/L 0.5mL,
It is 5.5 with NaOH solution tune pH value；NHS solution 90mmol/L 0.1mL；Derivative reagent amine compound solution 60mmol/L
0.35mL, with acetone or acetonitrile hydrotropy；The best coupling reaction time is 45 minutes after NHS solution is added.
Preferably, the derivative reagent amine compound is tryptamines.
The utility model has the advantages that small molecule monocarboxylic acid derivative-liquid phase chromatography detection method provided by the invention, more just compared with the prior art
Victory, sensitivity is higher, qualitative, quantitative is also more acurrate.
Detailed description of the invention
Fig. 1 is four kinds of organic acid derivatives liquid chromatograms；
Fig. 2 is formic acid derivates ultraviolet spectrogram；
Fig. 3 is acetogenin ultraviolet spectrogram；
Fig. 4 is propanoic derivatives ultraviolet spectrogram；
Fig. 5 is butanoic acid derivative ultraviolet spectrogram；
Fig. 6 is that MES optimizes additional amount selection figure；
Fig. 7 is that EDC optimizes additional amount selection figure；
Fig. 8 is that coupling agent NHS optimizes additional amount selection figure；
Fig. 9 is that amine compound optimizes additional amount selection figure；
Figure 10 is derivative reaction duration optimum choice figure；
Figure 11 is that 1 sample direct method of sample tests liquid chromatogram；
Figure 12 is that 1 analyte derivative method of sample tests liquid chromatogram；
Figure 13 is that 2 sample direct method of sample tests liquid chromatogram；
Figure 14 is that 2 analyte derivative method of sample tests liquid chromatogram；
Figure 15 is that 3 sample direct method of sample tests liquid chromatogram；
Figure 16 is that 3 analyte derivative method of sample tests liquid chromatogram；
Figure 17 is 20mg/L propionic acid standard sample ultraviolet spectrogram；
Figure 18 is ultraviolet spectrogram at propionic acid appearance in sample 3；
Figure 19 is 20mg/L formic acid standard sample ultraviolet spectrogram；
Figure 20 is ultraviolet spectrogram at formic acid appearance in sample 3；
Figure 21 is 20mg/L acetic acid standard sample ultraviolet spectrogram；
Figure 22 is ultraviolet spectrogram at acetic acid appearance in sample 3.
Above-mentioned sample 1 and sample 2 are fermenting crops straws liquid, and sample 3 is root exudates concentrate.
Specific embodiment
Embodiment 1
1, the preparation of reagent solution needed for derivative reaction
1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) solution: 30-60mmol/L
2- (N- morpholino) ethanesulfonic acid (MES) solution: 30-60mmol/L is 5-6.7 with NaOH solution tune pH value
N- hydroxysuccinimide (NHS) solution: 20-90mmol/L
Tryptamines solution: 30-60mmol/L is needed with acetone or acetonitrile hydrotropy.
2, standard solution is prepared
Prepare 4 kinds of monocarboxylic acid hybrid standard mother liquors are as follows: formic acid, acetic acid, propionic acid, butyric acid are 100mg/L.
3, derivative reaction
The derivative reaction of standard working curve sample: suitable 4 kinds of monocarboxylic acid hybrid standards mother liquor (0-1mL) is taken respectively
It is added in the scale test tube of 5mL, then is separately added into the MES solution of appropriate (0.2-1.0mL), 0.1-0.6mL EDC solution, 20
0.1-0.5mL NHS solution is added after minute, at this point, the pH value of reaction solution controls between 5.0-6.0, standing is reacted
After 0.5-1 hours, the tryptamines of appropriate (0.1-1.0mL) is added, stands reaction solution 10-20 hours, it is finally fixed with ultrapure water
Hold to 5mL, instrument is to be measured.Entire reaction process temperature is room temperature.The ratio of each compound is controlled in carboxylic acid in reaction system:
EDC:NHS (molar ratio)=1:(1-4): the range of (1-2), and carboxyl: amido ratio must be controlled between 2-4 (molar ratio),
Amido is excessive.
For the derivative reaction step of sample solution with the processing of standard solution, the sampling amount of sample solution can be according to sample
Situation takes (0.5-1.0mL).
In order to make derivatization operation become simpler, the sample injection bottle of 1.5mL can be used as reaction vessel, take identical
Mode of operation, sampling are all made of liquid-transfering gun and pipette, be finally settled to 1mL or supplied with liquid-transfering gun up to the ultrapure of 1mL total solution amount
Water, the additional amount of each reagent is referring to the 1/5 of the above-mentioned amount of pipetting, finally to hplc determination.
4, Instrument measuring
4.1 instrument condition
Using liquid chromatograph to after derivative reaction standard solution and sample solution be measured, with diode array
Detector carries out full wavelength scanner measurement, and quantitative detection wavelength is 223nm, and derivative liquid chromatogram is shown in that Fig. 1, each organic acid spread out
The ultraviolet spectrogram of biology is shown in Fig. 2-Fig. 5.
Hplc determination condition: mobile phase is acetonitrile+water；Chromatographic column be Agilent SB-C18,250mm ×
4.6mm, 5 μm, using acetonitrile (B) and water (A) as mobile phase, the flow velocity of 0.8mL/min, specific Gradient program is shown in Table 1.
1 organic acid derivatives liquid chromatogram of table tests Gradient program
4.2 derivative reaction condition optimizings
Derivative reaction can occur under given reaction condition in an experiment for the derivative reaction, but derivatization produces
The yield of object can have nothing in common with each other, therefore optimize to the concentration of derivative reagent each during derivative reaction, reaction condition, to obtain
Optimizing reaction conditions are obtained, reaches optimal derivatization products collection efficiency, is obtained in the case where not wasting reagent as far as possible optimal
Sensitivity.
Condition optimizing is changed in gradient with a certain condition in a series of experiments sample, and other conditions remain unchanged, and instrument is surveyed
Curve graph is made to peak area after fixed, the peak area of derivatization product is higher, then concentration is higher in solution, shows the derivative generated
Amount at most, can regard as optimal condition at this time.
4.2.1 the optimization additional amount of buffer MES
It is pipetted in 0.5mL to 5 5mL graded tube respectively from four kinds of monocarboxylic acid hybrid standard mother liquors of 100mg/L, each
Contain about 3.2 μm of ol of carboxyl in sample；Be separately added into again 60mmol/L MES solution (pH=5.5) 0.1,0.2,0.3,0.4,
0.5mL contains the MES of 6.0,12.0,18.0,24.0,30.0 μm of ol respectively；EDC 0.50mL (22.5 μ of 45mmol/L
Mol), it is separately added into the NHS solution (22.5 μm of ol) of 0.25mL 90mmol/L after twenty minutes；0.35mL is separately added into after 2 hours
60mmol/L (21 μm of ol) amine compound solution, then stands 10-20h, with ultrapure water constant volume and uses liquid chromatogram within second day
Instrument measurement.Each level is repeated 3 times, and takes its average value, as a result such as Fig. 6.
Known by Fig. 6, with the increase of MES additional amount, the concentration of formic acid derivates is gradually increased, and other several monocarboxylic acids
Derivatives concentration then first reduce and increase again, it is derivative when 30.0 μm of ol MES, the i.e. MES solution of 0.5mL 60mmol/L are added
The yield for changing product reaches preferable states, therefore selects the MES that 30 μm of ol are added.
4.2.2 the optimization additional amount of coupling reagent EDC
Operating process is similar to 4.2.1, pipettes 0.5mL respectively from four kinds of monocarboxylic acid hybrid standard mother liquors of 100mg/L
Into 5 5mL graded tubes, i.e., contain about 3.2 μm of ol of carboxyl in each sample；It is separately added into the MES solution of 60mmol/L again
(pH=5.5) 0.5mL (30 μm of ol)；The EDC 0.10 of 45mmol/L, 0.20,0.30,0.40,0.50,0.60mL, i.e., 9.0,
13.5,18.0,22.5,27.0μmol；It is separately added into the NHS solution of 0.1mL 90mmol/L (9 μm of ol) after twenty minutes；2 hours
After be separately added into 0.35mL 60mmol/L (21 μm of ol) amine compound solution, then stand 10-20h, use ultrapure water within second day
Constant volume simultaneously uses hplc determination.Each level is repeated 3 times, and takes its average value, as a result such as Fig. 7.
Known by Fig. 7, the EDC of addition is more, and the derivatization production concentration of generation is higher, but when additional amount is 22.5 μm of ol
After, second, third, the amount of butanoic acid derivative increase slowly, therefore the EDC additional amount of 22.5 μm of ol is optimal addn, but when knot
When closing the chromatogram complexity of liquid chromatogram measuring, EDC additional amount is more, and the more miscellaneous peak the stronger in chromatogram, therefore works as method
In the enough situations of sensitivity, may be selected less EDC additional amount, when this method selects EDC additional amount as 13.5 μm of ol compared with
It is good, sample analysis is neither influenced, and can guarantee the relatively easy of liquid chromatogram.The reality of liquid storage duration is prepared according to another EDC
Verify it is bright, EDC need to i.e. with i.e. use, otherwise be not susceptible to coupling reaction.
4.2.3 the optimization additional amount of coupling reagent NHS
Equally pipetted in 0.5mL to 5 5mL graded tube respectively from four kinds of monocarboxylic acid hybrid standard mother liquors of 100mg/L,
Contain about 3.2 μm of ol of carboxyl in each sample；It is separately added into MES solution (pH=5.5) 0.25mL (15 μ of 60mmol/L again
), mol the EDC 0.5mL (22.5 μm of ol) of 45mmol/L；Be separately added into after twenty minutes 90mmol/L NHS solution 0.1,0.2,
0.3,0.4,0.5mL, i.e., 9.0,18.0,27.0,36.0,45.0 μm of ol；0.35mL 60mmol/L (21 is separately added into after 2 hours
μm ol) amine compound solution, it then stands 10-20h, with ultrapure water constant volume and uses hplc determination within second day.Each
Level is repeated 3 times, and takes its average value, as a result such as Fig. 8.
By Fig. 8, when the NHS amount of addition is more, the peak area of derivatization product is smaller, and concentration is lower in ie in solution, therefore
Select the NHS additional amount of 9.0 μm of ol, i.e. 90mmol/L 0.1mL.
4.2.4 the optimization additional amount of derivative reagent amine compound (tryptamines)
Equally pipetted in 0.5mL to 5 5mL graded tube respectively from four kinds of monocarboxylic acid hybrid standard mother liquors of 100mg/L,
Contain about 3.2 μm of ol of carboxyl in each sample；It is separately added into MES solution (pH=5.5) 0.25mL (15 μ of 60mmol/L again
Mol), the EDC 0.5mL (22.5 μm of ol) of 45mmol/L, is separately added into the NHS solution 0.25mL of 90mmol/L after twenty minutes
(22.5 μm of ol) is separately added into 60mmol/L amine compound solution 0.15,0.25,0.35,0.45,0.55mL after 2 hours,
I.e. 9.0,15.0,21.0,27.0,33.0 μm of ol；10-20h is then stood, with ultrapure water constant volume and uses liquid chromatograph within second day
Measurement.Each level is repeated 3 times, and takes its average value, as a result such as Fig. 9.
Known by Fig. 9, under set reaction condition, when the additional amount of amine compound is 21.0 μm of ol, i.e.,
0.35mL, the peak area highest of the derivative products of each organic acid, i.e. concentration highest, that is, yield highest, therefore it is believed that this
Part is optimal conditions.
4.2.5 derivative reaction is time-optimized
Equally pipetted in 0.5mL to 5 5mL graded tube respectively from four kinds of monocarboxylic acid hybrid standard mother liquors of 100mg/L,
Contain about 3.2 μm of ol of carboxyl in each sample；It is separately added into MES solution (pH=5.5) 0.25mL (15 μ of 60mmol/L again
Mol), the EDC 0.5mL (22.5 μm of ol) of 45mmol/L, is separately added into the NHS solution 0.25mL of 90mmol/L after twenty minutes
60mmol/L amine compound solution 0.35mL (21 μ are added in (22.5 μm of ol) after 0,0.5,1.0,1.5,2.0 hour
Mol), then stand 10-20h, with ultrapure water constant volume and use hplc determination within second day.Each level is repeated 3 times, and takes it
Average value, as a result such as Figure 10.
As seen from Figure 10, formic acid derivates peak area reduces with the extension of derivative reaction time, small 0.5
Constantly peak area reaches peak value, illustrates the derivative reaction of formic acid quickly, therefore the optimal derivative reaction time of formic acid is 0.5
Hour；And for acetic acid, propionic acid and butyric acid, the peak area of derivatization product first increases with the extension of reaction time to drop afterwards
Low, when reaction reaches 1.0 hours, derivatization peak areas reaches highest, thus think 1 hour for three kinds of sour optimizations it is anti-
Between seasonable.Comprehensively consider, selects 0.75 hour (45 minutes) the derivatization optimization reaction duration sour as four kinds.
4.3 precision of method and accuracy
Precision of method is carried out to this method using optimizing reaction conditions and accuracy is tested, it is as a result as follows.
Calibration curve linear equation and linear correlation after organic acid standard sample is derivative are property such as table 2, as seen from Table 2,
Various organic acids resulting derivative curvilinear equation linear dependence after derivatization is R2> 0.99 can satisfy organic point
The requirement of analysis.The rate of recovery is shown in Table 3 after mark-on analyte derivative, by table 3, the recovery of standard addition range of four kinds of organic acids is 79.7%~
133%, in the controlled range of organic analysis, organic analysis requirement can be met.
2 organic acid derivatives calibrating curve equation of table and linear dependence
| Compound name | The range of linearity (mg/L) | Standard curve linear equation | Linearly dependent coefficient R2 |
| Formic acid derivates | 0,0.1~20.0 | Y=91633x+47861 | 0.9905 |
| Acetogenin | 0,0.1~20.0 | Y=232419x+43680 | 0.9957 |
| Propanoic derivatives | 0,0.1~20.0 | Y=244143x+92166 | 0.9963 |
| Butanoic acid derivative | 0,0.1~20.0 | Y=227003x+34280 | 0.9976 |
3 mark-on sample solution of table measures the rate of recovery after this method derivatization
Table 4 is (direct using organic acid derivative-liquid chromatography (derivatization method) and the direct liquid chromatography of sample solution
Method) survey timing method detection limit contrast table.As shown in Table 4, derivatization method not only detection limit at least 2 orders of magnitude lower than direct method,
And butyric acid can be measured, and do not absorbed under ultraviolet light using butyric acid when direct method, therefore the advantage of derivatization method is clearly.
Table 5 is then the method for the present invention --- method precision and instrument precision when derivatization method measures.It can be seen by table 5
Out, when low concentration (2mg/L) 4 kinds of organic acid derivatives method precision < 10%, each derivative instrument precision <
3%, and < 3%, repeatability is good, fully meets organic analysis for (10mg/L) each derivative method precision when high concentration
It is required that.
4 direct method of table and derivatization method detection limit comparison sheet
5 derivatization method method precision of table and instrument precision
| Title | Formic acid derivates | Acetogenin | Propanoic derivatives | Butanoic acid derivative |
| Method precision % (2mg/L, n=7) | 8.41 | 6.34 | 6.45 | 1.69 |
| Method precision % (10mg/L, n=7) | 2.13 | 2.40 | 2.56 | 2.86 |
| Instrument precision % (2mg/L, n=7) | 2.58 | 2.68 | 0.58 | 1.57 |
Note: in terms of peak area.
Table 6 is using result comparison sheet after derivatization method and Direct Determination same sample, and Figure 11-Figure 14 is sample 1 respectively
The liquid chromatogram tested with 2 direct method of sample and derivatization method, sample 1 and sample 2 are fermenting crops straws liquid.In direct method
In test, the retention time of formic acid is 2.748min, and appearance is forward, it is easy to by organic acids other in sample matrices or organic
The interference of compound, it is likely that be capped, it is difficult to qualitative and quantitative be carried out to it, the accuracy of measurement is influenced, if sample
Product matrix is fairly simple, then can be easier qualitative and quantitative.By Figure 11,12 it can be found that the retention time of acetic acid is
3.368min, appearance is obvious, but has the chromatographic peak of an organic acid or other organic matters in the front end of acetic acid chromatographic peak
It overlaps with it, the test result that this will cause acetic acid is relatively low, this result with two methods in table is than more consistent.Propionic acid
Retention time be 6.162min, chromatographic peak or so does not interfere with peak, therefore the test result of two methods is very close to still
The shortcomings that direct method, is: if the concentration of propionic acid is relatively low (direct method: propionate concentration < 5mg/L), that is, being less than in table 4 and provides
Propionic acid detection limit is then difficult to measure.Butyric acid can only can then be measured in derivatization method.
6 actual sample direct method of table and derivatization method test result comparison sheet
Note: sample 1 and 2 is fermenting crops straws liquid, and sample 3 is root exudates concentrate.
Therefore, from Figure 13 and Figure 14 as can be seen that derivatization method Interference Peaks near the derivative of formic acid and acetic acid are subtracted
Small, two kinds of sour qualitative and quantitatives are relatively more accurate.It, can also be by being adjusted in instrument analytic process if sample substrate is more complicated
Section eluent gradient elution program continues growing the separating degree between derivative and impurity peaks, with obtain it is more accurate it is qualitative with
It is quantitative.And for direct method, mobile phase is typically only capable to using phosphate buffer or acid solution as mobile phase, it is difficult to be carried out
Gradient elution, therefore separating effect is poor, as a result causes qualitative and quantitative inaccurate.For propionic acid, derivatization method substantially increases propionic acid
Sensitivity, and propanoic derivatives, nearby without other Interference Peaks, qualitative, quantitative is not accurate and reliable.Butyric acid does not almost have ultraviolet light
There is absorption, so direct method can not measure butyric acid；But derivatization method can test it in high sensitivity, and butanoic acid derivative is attached
Nearly noiseless peak, so the qualitative and quantitative of butyric acid is accurate and reliable.
Sample 3 is the concentrating sample of root exudates in table 6, is found after comparison, and two methods measure propionic acid and fourth
The result of acid meets expection, because propionic acid concentration when derivatization method measures is lower, the detection lower than direct method is limited, therefore in direct method
In be to be not detected.Although can integrate to obtain a chromatographic peak at the appearance time of propionic acid shown in figure 15, through ultraviolet light
Discovery is not the chromatographic peak of propionic acid after spectrogram judgement, sees that Figure 17 and Figure 18, Figure 18 indicate at the time appearance be third
The uv absorption spectra of acid, it should be fluctuation caused by baseline noise, this easily causes false positive test results in analysis.
Go out peak position to formic acid in Figure 15 (sample 3) by retention time to be integrated to obtain apparent formic acid chromatography
Peak；But in derivatization method, i.e. the chromatographic peak of formic acid derivates is then hardly visible in Figure 16；Therefore, again to the " first of Figure 15
The ultraviolet spectrogram (Figure 19-20) of formic acid chromatographic peak carries out ultraviolet spectrogram comparison, discovery in sour chromatographic peak " and formic acid standard sample
Ultra-violet absorption spectrum is had any different, and Figure 15 ultraviolet spectrogram explanation should have absorption (to have at 280nm ultraviolet light containing other groups
Absorb), showing the peak at least should not be pure formic acid peak.This original for causing formic acid result higher when should be Direct Determination
Cause.
It is found after Figure 21 is compared with Figure 22, the ultraviolet spectra of acetic acid shows the absorption peak of other compounds in Figure 22.
But having with previous compound chromatographic peak overlapping in 3 liquid chromatogram of sample at the appearance of acetic acid, this should be second in sample
The uv absorption spectra of acid and the discrepant reason of uv absorption spectra of acetic acid standard sample；And this period have it is more
A overlap of peaks, baseline is high, and will cause acetic acid concentration can be relatively low, so direct method measures Lower result than derivatization method.
5 conclusions
Several monocarboxylic acid derivative-liquid phase chromatography detection methods proposed by the present invention are accurate with qualitative, quantitative, remolding sensitivity is straight
Connection improves at least two order of magnitude, and can measure butyric acid, and advantage is clearly.
Claims (1)
1. a kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method, it is characterised in that step are as follows:
Chromatographic condition: quantitative detection wavelength is 223 nm, and chromatographic column is Agilent SB-C18, and column oven temperature is 35 DEG C；With
Acetonitrile and water are as mobile phase, the fixed flow rate of 0.8 mL/min；Gradient elution program are as follows: acetonitrile volume ratio is at 0 minute
EP (end of program) when being reduced to 10%, 34 minutes when rising to 70%, 32 minutes when 10%, 30 minutes again;
The preparation of reagent solution needed for derivative reaction: 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate
45 mmol/L of solution；2-(N- morpholino) 60 mmol/L of ethanesulfonic acid MES solution is 5-6.7 with NaOH solution tune pH value；N-
Hydroxysuccinimide NHS solution 90mmol/L；60 mmol/L of tryptamines solution, with acetone or acetonitrile hydrotropy；
Standard solution is prepared: preparing 4 kinds of monocarboxylic acid hybrid standard mother liquors are as follows: formic acid, acetic acid, propionic acid, butyric acid are 100 mg/L；
Derivative reaction:
The derivative reaction of standard solution: take 4 kinds of monocarboxylic acid hybrid standard mother liquors of 0-1 mL that the scale examination of 5 mL is added respectively
Guan Zhong, then it is separately added into the 2- of 0.5 mL 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt acid salt solution, 0.5mL
The pH value control of 0.1 mL N- hydroxysuccinimide solution, reaction solution is added in (N- morpholino) ethanesulfonic acid solution after twenty minutes
System is between 5.5, after standing reaction 0.75 hour, adds the tryptamines solution of 0.35 mL, stands reaction solution 10-20 hours,
It finally is settled to 5mL with ultrapure water, instrument is to be measured；Entire reaction process is completed at room temperature, carboxylic acid in reaction system:
1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate: N- hydroxysuccinimide molar ratio=1:1 ~ 4:1 ~ 2
Range, carboxyl: the molar ratio of tryptamines is between 2-4；
The derivative reaction of sample solution: derivative reaction of the step with standard solution, sampling amount 0.5-1.0 mL；
Assay: precision draws the standard solution and each 20 μ L of sample solution after derivative reaction, injects liquid chromatograph,
Measurement is to get result.
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