CN108562670A - A kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method - Google Patents

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 is：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.Several monocarboxylic acids proposed by the present invention derive liquid phase chromatography detection method, and with qualitative, quantitative, accurate, remolding sensitivity direct method improves at least two order of magnitude, and can measure butyric acid, and advantage is clearly.

Description

A kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method

Technical field

The invention belongs to a kind of organic acid detection techniques of the related fields such as agricultural, ecology and environment, more particularly to ecology The highly sensitive Instrumental Analysis of several low content monocarboxylic acids and detection in ambient water sample, plant rhizosphere sample.

Background technology

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, and to pass through the complex mechanisms such as chelating and ligand exchange influence 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 influence, 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 To include 2 major class of aliphatic and aromatic series, main aromatic organic acid (also commonly referred to as allelochemical) 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 Include mainly：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 the adverse effect that brings, such as several aliphatic monocarboxylic acids in plant rhizosphere --- formic acid, lactic acid, acetic acid, propionic acid, Butyric acid etc., amount appropriate has certain positive influences, but rotten of plant etc. is likely to result in after content increases to certain value, It in turn results in and hinders the harmful effects such as plant growth；In addition, the content of these types of organic acid is usually relatively low, instrument detection sensitivity Difference, measurement difficulty is larger, therefore must have 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 that one kind with fastest developing speed in recent years is divided 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 compared to conventional method with capillary electrophoresis (CE/UV), the sensitivity of detection.

GC/FID methods or GC/MS methods measure organic acid, and separating effect is generally fine on a column for sample, but to sample Pre-treatment is complex to be taken, and sample needs to examine by complicated derivatization treatment (usually esterification or silanization etc.) It surveys, this can cause the loss of object and reduce the rate of recovery to a certain extent, precision, more less reproducible, simultaneously because The introducing of derivatization reagent, chromatogram also can be more complicated.

HPLC methods 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 can influence object It surveys, is difficult then to measure for low content sample.If using dedicated organic acid measuring column, chromatographic column is expensive, and right 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 methods analyze 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 rules It is now that sample dosage is seldom, analysis time is shorter, and interference is small compared with HPLC methods and IC methods, but the stability ratio HPLC methods and IC of instrument Method is more poor, and for low content organic acid sample, there is also the low problems of detection sensitivity, and operating cost is also relatively high.

Invention content

The technical issues of solution：The present invention is directed to 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：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 is：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： Preparing 4 kinds of monocarboxylic acid hybrid standard mother liquors is：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 solutions, 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 ultra-pure water, instrument is to be measured；Entire reaction process It completes at ambient temperature, carboxylic acid in reaction system:EDC:Molar ratio=1 NHS:(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：Step is the same as the derivative reaction of standard solution, sampling Measure 0.5-1.0mL；Assay：Standard solution after precision absorption derivative reaction and each 20 μ L of sample solution, inject liquid phase Chromatograph measures to get result.

The optimization of each derivatization reagent is a concentration of：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.

Advantageous effect：Small molecule monocarboxylic acid derivative-liquid phase chromatography detection method provided by the invention, more just compared with the prior art Victory, sensitivity higher, qualitative, quantitative are also more acurrate.

Description of the drawings

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 addition selection figure；

Fig. 7 is that EDC optimizes addition selection figure；

Fig. 8 is that coupling agent NHS optimizes addition selection figure；

Fig. 9 is that amine compound optimizes addition 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 spectrograms；

Figure 18 is ultraviolet spectrogram at propionic acid appearance in sample 3；

Figure 19 is 20mg/L formic acid standard sample ultraviolet spectrograms；

Figure 20 is ultraviolet spectrogram at formic acid appearance in sample 3；

Figure 21 is 20mg/L acetic acid standard sample ultraviolet spectrograms；

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 implementation mode

Embodiment 1

1, the preparation of the reagent solution needed for derivative reaction

1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC) solution：30-60mmol/L

2- (N- morpholinoes) ethanesulfonic acid (MES) solution：30-60mmol/L is 5-6.7 with NaOH solution tune pH value

N- hydroxysuccinimides (NHS) solution：20-90mmol/L

Tryptamines solution：30-60mmol/L needs to use acetone or acetonitrile hydrotropy.

2, standard solution is prepared

Preparing 4 kinds of monocarboxylic acid hybrid standard mother liquors is：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 solutions, 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 ultra-pure 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 is taken (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 up to the ultrapure of 1mL total solution amounts with liquid-transfering gun The addition of water, each reagent finally waits for hplc determination with reference to the 1/5 of the above-mentioned amount of pipetting.

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 be optimized to the concentration of each derivative reagent, reaction condition during derivative reaction, to obtain Optimizing reaction conditions are obtained, reaches best derivatization products collection efficiency, is obtained in the case where not wasting reagent 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 is most, can regard as optimal condition at this time.

4.2.1 the optimization addition of buffer solution MES

It is pipetted respectively in 0.5mL to 5 5mL graded tube 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；It is separately added into 0.35mL after 2 hours 60mmol/L (21 μm of ol) amine compound solution, then stands 10-20h, with ultra-pure water constant volume and uses liquid chromatogram within second day Instrument measures.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 additions, the concentration of formic acid derivates gradually increases, 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 addition of coupling reagent EDC

Operating process is similar to 4.2.1, and 0.5mL is pipetted 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 ultra-pure 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 addition is 22.5 μm of ol After, second, third, the amount of butanoic acid derivative increase slow, therefore the EDC additions of 22.5 μm of ol are optimal addn, but when knot When closing the chromatogram complexity of liquid chromatogram measuring, EDC additions are more, and the more miscellaneous peak the stronger in chromatogram, therefore work as method In the case that sensitivity is enough, may be selected less EDC additions, this method select EDC additions for 13.5 μm of ol when compared with It is good, sample analysis is neither influenced, and can ensure the relatively easy of liquid chromatogram.The reality that liquid stores 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 addition of coupling reagent NHS

Equally pipetted respectively in 0.5mL to 5 5mL graded tube 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 are separately added into after 2 hours μm ol) amine compound solution, it then stands 10-20h, with ultra-pure 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 amounts of addition are more, the peak area of derivatization product is smaller, and concentration is lower in ie in solution, therefore Select the NHS additions of 9.0 μm of ol, i.e. 90mmol/L 0.1mL.

4.2.4 the optimization addition of derivative reagent amine compound (tryptamines)

Equally pipetted respectively in 0.5mL to 5 5mL graded tube 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, are 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 compounds 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 ultra-pure water constant volume and uses liquid chromatograph within second day It measures.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 addition 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 respectively in 0.5mL to 5 5mL graded tube 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, are 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 ultra-pure 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 drops afterwards as the extension in reaction time first increases 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.Consider, selects the derivatization optimization reaction duration that 0.75 hour (45 minutes) are 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 derivative are property such as table 2, as seen from Table 2, Various organic acids derivative curvilinear equation linear dependence of gained after derivatization is R²＞ 0.99 can meet 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 ranging from 79.7% of four kinds of organic acids~ 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 when derivatization method measures and instrument precision.It can be seen by table 5 Go out, the method precision of (2mg/L) 4 kinds of organic acid derivatives is equal when low concentration<10%, each derivative instrument precision is equal< 3%, and (10mg/L) each derivative method precision is equal when high concentration<3%, repeatability is good, fully meets organic analysis 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 results contrast table 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 other organic acids 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 measured 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 there are one organic acids or the chromatographic peak of other organic matters in the front end of acetic acid chromatographic peak It overlaps with it, this can cause the test result of acetic acid relatively low, this is consistent with the results contrast of two methods in table.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 relatively low (the direct method of the concentration of propionic acid：Propionate concentration<5mg/L), that is, it is less than in table 4 and provides Propionic acid detection limit is difficult then 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 so that Interference Peaks subtract near the derivative of formic acid and acetic acid Small, two kinds of sour qualitative and quantitatives are relatively more accurate.It, can also be by being adjusted during Instrumental Analysis 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 relatively low, the detection less than direct method limits, 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 judges, 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 Sour chromatographic peak " and the ultraviolet spectrogram (Figure 19-20) of formic acid chromatographic peak in formic acid standard sample carry out ultraviolet spectrogram comparison, find Ultra-violet absorption spectrum is had any different, and Figure 15 ultraviolet spectrograms explanation, which should contain other groups, has absorption (to have at 280nm ultraviolet light Absorb), show the peak at least and 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 discrepant reason of uv absorption spectra of the uv absorption spectra and acetic acid standard sample of acid；And this period have it is more A overlap of peaks, baseline is high, acetic acid concentration can be caused 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 (3)

1. a kind of small molecule monocarboxylic acid derivative-liquid phase chromatography detection method, it is characterised in that step is：

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 is：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：EDC solution 30-60 mmol/L；MES solution 30-60 mmol/L are used NaOH solution tune pH value is 5-6.7；NHS solution 20-90mmol/L；Derivative reagent amine compound solution 30-60 mmol/L, With acetone or acetonitrile hydrotropy；

Standard solution is prepared：Preparing 4 kinds of monocarboxylic acid hybrid standard mother liquors is：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 MES solution of 0.1-0.6 mL EDC solutions, 0.2-1.0 mL, 0.1-0.5 mL are added after twenty minutes NHS solution, reaction solution pH value control between 5.0-6.0, stand reaction 0.5-1 hours after, add 0.1-1.0 mL Derivative reagent amine compound solution, stand reaction solution 10-20 hours, be finally settled to 5mL with ultra-pure water, instrument waits for It surveys；Entire reaction process is completed at ambient temperature, carboxylic acid in reaction system:EDC:Molar ratio=1 NHS:（1~4）:（1~2）'s Range, carboxyl:The molar ratio of amido substance is between 2-4；

The derivative reaction of sample solution：Step is the same as the derivative reaction of standard solution, sampling amount 0.5-1.0 mL；

Assay：Standard solution after precision absorption derivative reaction and each 20 μ L of sample solution, inject liquid chromatograph, It measures to get result.

2. small molecule monocarboxylic acid derivative-liquid phase chromatography detection method according to claim 1, it is characterised in that each derivatization reagent Optimization it is a concentration of：45 mmol/L of EDC solution, 0.5 mL；60 mmol/L of MES solution, 0.5 mL, with NaOH solution tune pH value It is 5.5；90 mmol/L 0.1mL of NHS solution；60 mmol/L 0.35mL of derivative reagent amine compound solution, with acetone or Acetonitrile hydrotropy；The best coupling reaction time is 45 minutes after NHS solution is added.

3. small molecule monocarboxylic acid derivative-liquid phase chromatography detection method according to claim 1, it is characterised in that the derivative reagent Amine compound is tryptamines.