CN102507817B - Method for forecasting leading chromatographic peak shape under multi-order programmed temperature condition - Google Patents

Abstract

The invention discloses a method for predicting the peak shape of tongue protruding chromatogram under the condition of multi-stage temperature programming. The method mainly includes: calculating the chromatographic retention factor values at different temperatures according to the virtual dead time; using a computer program to search for the peak shape parameter value psp under constant temperature conditions according to the nonlinear plate theory; Retention factor and peak shape parameters, and regression analysis to establish the functional relationship between the two and temperature; set the temperature rise program, and obtain the predicted tongue-out chromatographic peak shape through computer operations, and compare it with the experimental peak shape obtained under the same programmed temperature rise conditions. shape for comparison. The invention provides an important basis for the qualitative determination of such chromatographic peaks as protruding tongue peaks, optimizes the chromatographic separation conditions, and simultaneously provides a new approach for the accurate quantification of common overlapping chromatographic peaks in laboratories. In addition, the invention has a wide application range and high prediction accuracy.

Description

A Method for Predicting the Peak Shape of Tongue-out Chromatography under Multi-step Temperature Programmable Conditions

technical field

The invention relates to a method for predicting the peak shape of a tongue protruding chromatogram under the condition of multi-stage temperature programming, and belongs to the technical field of gas chromatography.

Background technique

In the field of chromatographic analysis, the prediction of chromatographic peak shape is a very important research content. For complex compounds with a wide range of boiling points and compositions, it is necessary to use temperature programming conditions to achieve separation between components. If based on the retention time and peak shape parameter data of a small number of samples, with the help of computer programs, the prediction of the chromatographic peak shape under the condition of programmed temperature rise can be realized, not only the accurate prediction of the retention time can be realized, the chromatographic separation conditions can be optimized, and the chromatographic qualitative can be provided. Can provide new avenues for accurate quantification of overlapping peaks. Therefore, the accurate prediction of chromatographic peak shape, especially the prediction of chromatographic peak shape under programmed temperature conditions, has great innovative and practical value.

At present, the work on the prediction of chromatographic peak shape under constant temperature conditions has been carried out at home and abroad, but the work on the prediction of chromatographic peak shape under programmed temperature conditions has not been reported.

Contents of the invention

The purpose of the present invention is to provide a method based on the nonlinear tray theory to predict the peak shape of the tongue-out chromatogram under the condition of multi-stage temperature programming. Tongue chromatographic peak shape, and the prediction process is simple;

The present invention is achieved through the following technical scheme, a method for predicting the peak shape of the tongue-out chromatogram under the condition of multi-stage temperature programming, the method is based on the nonlinear tray theory, and is aimed at HP-5 chromatographic column (hereinafter referred to as chromatographic column), adopts arbitrary constant virtual dead time in the prediction process, is characterized in that comprising following process:

1) Setting of virtual dead time τ: The temperature range of the chromatographic column setting is 30-250°C, and the measured compound is measured at T ₁ =30°C, T ₂ =50°C, T ₃ =100°C, T ₄ =150°C ℃, T ₅ ＝200 ℃ and T ₆ ＝250 ℃ under six constant temperatures of retention time t _R1 , t _R2 , t _R3 , t _R4 , t _R5 and t _R6 , determine the value of the minimum retention time, if it is less than the minimum retention time Any time value of the time value can be used as a virtual dead time;

2) Calculation of the retention factor k under constant temperature conditions: according to the six retention times t _R1 , t _R2 , t _R3 , t _R4 , t _R5 , t _R6 determined in step 1) and the virtual dead time τ under constant temperature, Using formula 1, calculate the corresponding retention factors at six constant temperatures: k ₁ , k ₂ , k ₃ , k ₄ , k ₅ and k ₆ ,

k＝(t _R -τ)/τ Formula 1

In formula 1: k is the retention factor,

τ is the virtual dead time,

t _R is the retention time corresponding to each temperature point;

3) Calculation of peak shape parameter (psp) under constant temperature conditions:

The peak shape parameter psp is defined as the coefficient in the functional relationship between the concentration of the test compound on the same tray between the stationary phase and the mobile phase established based on the nonlinear tray theory, and the functional relationship is as follows:

$C_S=\mathrm{psp}\·C_m^2+(k+1)\·C_m$ Formula 2

C _S +C _M ＝C _n Formula 3

In formula 2: psp is the peak shape parameter value;

C _S , C _M are the concentrations of the compounds to be tested in the stationary phase and mobile phase on the same tray;

In formula 3: _C is the sum of the concentration of the compound to be tested in the stationary phase and the mobile phase on the same tray;

Run the following computer program to calculate the peak shape parameter values under different temperature conditions:

Firstly, input the known parameters in the program: the chromatographic column temperature T under constant temperature conditions, the initial concentration C _M00 of the compound to be tested, the retention factor k of the compound to be tested at this temperature condition, the number of theoretical plates N and the number of jumps l; Using Equation 2 and Equation 3, input three different peak shape parameter values, compare the peak shape obtained by computer program operation with the experimental peak shape, and obtain the relationship between the peak shape parameter and the peak shape size; constantly adjust the input The size of the peak shape parameter value, until the obtained peak shape under the constant temperature condition completely matches the experimental peak shape, this value is the actual peak shape parameter value of the compound to be tested under the temperature condition;

Using the retention factor _ki corresponding to the i-th jump, and the set peak shape parameter value psp, according to formula 4 and formula 5, respectively calculate the compounds to be tested in blocks 1, 2, 3...n...N Concentrations of stationary and mobile phases in the trays:

$C_{\mathrm{Sni}}=\mathrm{psp}\·C_{\mathrm{Mni}}^2+(k+1)\·C_{\mathrm{Mni}}$ Formula 4

C _Sni + C _M ni = C _ni formula 5

In formula 4: C _Sni and C _Mni are the concentration of the compound to be tested in the stationary phase and mobile phase in the nth plate when the number of jumps is i respectively;

In formula 5: C _ni is when the number of jumps is i, the total concentration of the compound to be tested in the nth column plate, which is determined by formula 6:

C _ni ＝C _S(n-1)i +C _M(n-1)(i-1) Formula 6

C _M00 = 1 μg/mL

In Formula 6: C _M00 is the initial concentration of the compound to be tested,

C _S(n-1)i is the concentration of the compound to be tested in the stationary phase of the n-1th column plate when the number of jumps is i,

C _M(n-1)(i-1) is the concentration of the compound to be tested in the mobile phase of the n-1th plate when the number of jumps is i-1;

When the compound to be tested jumps to the last tray, the compound to be tested is still distributed according to the relationship between formula 4 and formula 5. When the next jump is performed, the compound to be tested in the mobile phase on the last tray flows out, and the moment is recorded The concentration value of the effluent, the test compound in the stationary phase and the test compound brought in by the mobile phase of the previous plate are redistributed, so that until the set number of jumps is reached, all the test compounds are guaranteed to flow out of the chromatographic column, thus Obtain the data points corresponding to the outflow concentration value of the compound to be tested at each jump, and the graph formed by these data points is the predicted tongue-out chromatographic peak shape;

Set six temperature values of T ₁ , T ₂ , T ₃ , T ₄ , T ₅ and T ₆ in the above computer program, and obtain corresponding peak shape parameter values psp ₁ , psp ₂ , psp ₃ , psp _4. psp ₅ and psp ₆ ;

4) Determine the corresponding temperature T _i when the compound to be tested undergoes any jump in the chromatographic column under the condition of programmed temperature rise:

(1) Determine the time Δτ required for each jump by Equation 6,

Δτ=τ/(N-1) Equation 6

In formula 6: τ is the virtual dead time, which has been determined by step 1),

N is the inherent theoretical plate number of the chromatographic column;

(2) The compound to be tested jumps i times in the chromatographic column, a total time t _i is required, calculated by formula 7,

t _i =i×Δτ Formula 7

In formula 7, i is the number of jumps;

(3) In the multi-stage temperature program, calculate the total time t of the multi-stage temperature program:

t＝t _h1 +t ₁ +t _h2 +t ₂ Formula 8

In formula 8: t _h1 is the holding time of the initial temperature, the empirical value is: 1-5min,

t _h2 is the holding time of the end temperature of the first stage of programmed temperature rise, the empirical value is: 1-5min,

t ₁ is the time required for the first stage of temperature programming,

t ₂ is the time required for the second stage of temperature programming,

t ₁ and t ₂ are calculated by formula 6 and formula 7 respectively:

t ₁ =(T _m -T ₀ )/r ₁ Formula 9

t ₂ =(T _f -T _m )/r ₂ Formula 10

In formula 9: T _m is the termination temperature of the first stage of temperature programming,

T ₀ is the starting temperature,

r ₁ is the heating rate of the first stage of programmed heating;

In formula 10: T _f is the termination temperature of the second stage temperature programming,

r ₂ is the heating rate of the second stage of temperature programming,

Among them, the empirical value range of r ₁ and r ₂ is 5-30°C/min;

(4) Determine the temperature T _i corresponding to the chromatographic column at the time of the i-th jump of the compound to be tested:

When t _i <t _h1 , the column temperature T _i =T ₀ ,

When t _h1 <t _i <(t _h1 +t ₁ ), then column temperature T _i =r ₁ ×(t ₁ -t _h1 )+T ₀ ,

When (t _h1 +t ₁ )≤t _i ≤(t _h1 +t ₁ +t _h2 ), then column temperature T _i =T _m ,

When (t _h1 +t ₁ +t _h2 )<t _i <t, then column temperature T _i =r ₂ ×(t _i -t ₁ -t _h1 -t _h2 )+T _m ,

When t _i >t, the column temperature T _i =T _f ;

5) Determination of the relationship between retention factor k and peak shape parameter psp and temperature under multi-stage temperature programming conditions:

(1) Using the six constant temperature values T ₁ , T ₂ , T ₃ , T ₄ , T ₅ and T ₆ and the corresponding retention factors k ₁ , k ₂ , k ₃ , k ₄ , k ₅ and k ₆ , through regression analysis, the functional relationship between retention factor k and temperature T is obtained as formula 11:

lnk＝aT ³ +bT ² +cT+d Formula 11

Among them, parameters a, b, c and d are fixed values;

From this, the retention factor k _i corresponding to any temperature point T _i in the temperature program is calculated;

(2) Using six constant temperature values T ₁ , T ₂ , T ₃ , T ₄ , T ₅ and T ₆ and the corresponding peak shape parameter values psp ₁ , psp ₂ , psp ₃ , psp ₄ obtained in step 3) , psp ₅ and psp ₆ , through regression analysis, the functional relationship between the peak shape parameter psp and the temperature T is obtained as formula 12:

ln psp=a'T ³ +b'T ² +c'T+d' Formula 12

In formula 12, the parameters a', b', c' and d' are all fixed values;

From this, the peak shape parameter psp corresponding to any temperature point T _i in the temperature program is calculated;

6) Obtaining the predicted peak shape of the compound to be tested under the condition of multi-stage temperature programming:

Enter the following data into the computer program: column phase β, initial concentration of the compound to be tested C _M00 , number of theoretical plates N, number of jumps n, six constant temperature values, virtual dead time τ, retention factor and peak shape parameters The parameters in the relationship with temperature, the initial temperature of the multi-stage temperature program, the temperature at the end of the first stage, the temperature at the end of the second stage, the heating rate of the first stage, the heating rate of the second stage, the temperature maintained at the initial temperature Time and the time maintained at the end temperature of the first stage program temperature increase; through the computer operation program described in step 3), the predicted tongue-out chromatographic peak shape of the compound to be tested under the temperature program temperature condition is obtained.

The present invention has the advantages of realizing the prediction of the protruding tongue peak shape under the condition of multi-stage temperature programming, providing an important basis for the qualitative determination of the chromatographic peak shape of the protruding tongue peak, optimizing the chromatographic separation conditions, and simultaneously analyzing the common The accurate quantification of overlapping chromatographic peaks provides a new way; in the calculation process, it is not necessary to accurately determine the value of the dead time, and a certain value of the dead time can be set arbitrarily, so the prediction process is very simple; the temperature points taken in the prediction process are many, The range of variation is large, and the peak shape of the tongue protruding chromatogram can be better predicted in a wide range of temperature variation. This method has a wide range of applications and high prediction accuracy.

Description of drawings

Fig. 1 is the computer operation flowchart of the method for predicting the peak shape of the tongue protruding chromatogram under the condition of multi-stage temperature programming in the present invention.

FIG. 2 is a comparison chart of the experimental peak shape and the predicted peak shape under the temperature rising program condition of A in Example 1. The solid line in the figure represents the predicted peak shape, and the dashed line represents the experimental peak shape.

FIG. 3 is a comparison chart of the experimental peak shape and the predicted peak shape under the B heating program condition in Example 1. The solid line in the figure represents the predicted peak shape, and the dashed line represents the experimental peak shape.

FIG. 4 is a comparison chart of the experimental peak shape and the predicted peak shape under the temperature rising program condition C in Example 1. The solid line in the figure represents the predicted peak shape, and the dashed line represents the experimental peak shape.

Fig. 5 is a comparison chart of the experimental peak shape and the predicted peak shape under the condition of the heating program A in Example 2. The solid line in the figure represents the predicted peak shape, and the dashed line represents the experimental peak shape.

Fig. 6 is a comparison chart of the experimental peak shape and the predicted peak shape under the B heating program condition in Example 2. The solid line in the figure represents the predicted peak shape, and the dashed line represents the experimental peak shape.

Fig. 7 is a comparison chart of the experimental peak shape and the predicted peak shape under the condition of C heating program in Example 2. The solid line in the figure represents the predicted peak shape, and the dashed line represents the experimental peak shape.

Detailed ways

Example 1

Instruments: HP6890 gas chromatograph, hydrogen flame ionization detector, 6890 gas chromatograph workstation;

Chromatographic column: non-polar HP-5 (5% phenylmethylpolysiloxane) column;

Conditions: The temperature of the detector is 250°C, and the temperature of the injection port is 250°C;

Carrier gas: use high-purity nitrogen gas (purity not less than 99.999%), constant flow operation mode, that is, the carrier gas is at the outlet of the column,

The mass flow rate was kept constant at 1mL/min;

Injection method: split injection, the split ratio is 50:1, each injection volume is 0.2 μL, and the concentration is 1 μg/mL;

(1) Heptanoic acid was selected as the compound to be tested, and its retention times at six constant temperatures of 30°C, 50°C, 100°C, 150°C, 200°C and 250°C were measured on the HP-5 column, which were 25.01min, 14.32min, 7.30min, 3.07min, 2.34min and 2.08min;

(2) Take the virtual dead time τ=1.85min, and calculate the retention factors of heptanoic acid at six constant temperatures according to formula 1, which are respectively: 2.53, 1.91, 1.08, -0.42, -1.33 and -2.12. At lower temperatures, the distribution of heptanoic acid between the two phases is relatively slow, and is affected by mass transfer resistance diffusion items, etc. Therefore, when using Equation 2 to fit the curves of the relationship between retention factor and temperature at six constant temperatures , the above parameters need to be corrected, and finally the parameters a, b, c and d are obtained, which are: -8.4224×10 ^-7 , 1.2051×10 ^-3 , -0.5881 and 96.8190; In , the relationship between retention factor and temperature is as follows:

lnk＝-8.4224×10 ^-7 T ³ +1.2051×10 ^-3 T ² -0.5881T+96.8190

(3) First input the following known parameters into the computer program: constant temperature T ₁ (30°C), initial concentration of the compound to be tested 1 μg/mL, retention factor k ₁ (2.53), number of theoretical plates N and number of jumps l; Constantly adjust the value of the input peak shape parameter, so that the final predicted peak shape is completely consistent with the experimental peak shape, and the peak shape parameter value of heptanoic acid at 30°C is 21.00.

Similarly, the peak shape parameters of heptanoic acid at 50°C, 100°C, 150°C, 200°C, and 250°C can be obtained as 16.97, 7.00, 2.00, -1.83, and -9.21, respectively.

Through regression analysis, draw heptanoic acid under the condition of multi-stage temperature programming, the relational expression of peak shape parameter and temperature is as follows:

ln psp＝-4.4509×1 ^-6 T ³ +5.8055×10 ^-3 T ² -2.5711T+391.7654

Choose from three different temperature programs, which are:

A program temperature rise 30°C (hold 2min) → 5°C/min → 70°C (hold 1min) → 25°C/min → 250°C;

B Program temperature rise 30°C (hold for 2min)→10°C/min→70°C (hold for 1min)→25°C/min→250°C;

C program temperature rise 30°C (hold for 2min)→15°C/min→70°C (hold for 1min)→25°C/min→250°C;

Enter the following data into the computer program: column phase β, initial concentration of the compound to be tested C _M00 , number of theoretical plates N, number of jumps n, six constant temperature values, virtual dead time τ, retention factor and peak shape parameters The parameters in the relationship with temperature, the initial temperature of the multi-stage temperature program, the temperature at the end of the first stage, the temperature at the end of the second stage, the heating rate of the first stage, the heating rate of the second stage, the temperature maintained at the initial temperature Time and the time maintained under the first-stage temperature program temperature termination temperature; run the computer program to obtain the predicted chromatographic peak shapes of heptanoic acid under the above-mentioned three temperature program temperature conditions respectively.

(4) Use the HP-5 column to obtain the corresponding chromatographic peak shapes of heptanoic acid under the above three programmed temperature conditions on the gas chromatograph, and compare them with the predicted peak shapes, as shown in Figures 2, 3, and 4 in turn.

It can be seen from the above respective comparison charts that the predicted peak shape has reached a high degree of agreement with the experimental peak shape, which confirms the accuracy of predicting the tongue-out chromatographic peak shape under different temperature programming conditions based on the nonlinear tray theory. the feasibility of the method.

In this embodiment, the prediction relative error %=(prediction result−experimental result)/experimental result×100. The results are shown in Table 1:

Table 1 The relative error between the predicted peak shape of heptanoic acid and the experimental peak shape under different programmed temperature conditions

Example 2

Experimental apparatus, chromatographic column, condition, carrier gas and sample injection mode are the same as embodiment 1;

(1) Heptanoic acid was selected as the compound to be tested, and its retention times at six constant temperatures of 30°C, 50°C, 100°C, 150°C, 200°C and 250°C were measured on the HP-5 column, which were 25.01min, 14.32min, 7.30min, 3.07min, 2.34min and 2.08min;

(2) Take the virtual dead time τ=1.85min, and calculate the retention factors of heptanoic acid at six constant temperatures according to formula 1, which are respectively: 2.53, 1.91, 1.08, -0.42, -1.33 and -2.12. At lower temperatures, the distribution of heptanoic acid between the two phases is relatively slow, and is affected by mass transfer resistance diffusion items, etc. Therefore, when using Equation 2 to fit the curves of the relationship between retention factor and temperature at six constant temperatures , the above parameters need to be corrected, and finally the parameters a, b, c and d are obtained, which are: -8.4224×10 ^-7 , 1.2051×10 ^-3 , -0.5881 and 96.8190; In , the relationship between retention factor and temperature is as follows:

lnk＝-8.4224×10 ^-7 T ³ +1.2051×10 ^-3 T ² -0.5881T+96.8190

(3) First input the following known parameters into the computer program: constant temperature T ₁ (30°C), initial concentration of the compound to be tested 1 μg/mL, retention factor k ₁ (2.53), number of theoretical plates N and number of jumps l; Constantly adjust the value of the input peak shape parameter, so that the final predicted peak shape is completely consistent with the experimental peak shape, and the peak shape parameter value of heptanoic acid at 30°C is 21.00.

Similarly, the peak shape parameters of heptanoic acid at 50°C, 100°C, 150°C, 200°C, and 250°C can be obtained as 16.97, 7.00, 2.00, -1.83, and -9.21, respectively.

Through regression analysis, draw heptanoic acid under the condition of multi-stage temperature programming, the relational expression of peak shape parameter and temperature is as follows:

ln psp＝-4.4509×10 ^-6 T ³ +5.8055×10 ^-3 T ² -2.5711T+391.7654

Choose from three different temperature programs, which are:

A program temperature rise 70°C (hold 2min) → 15°C/min → 150°C (hold 1min) → 25°C/min → 250°C;

B Program temperature rise 70°C (hold for 2min)→20°C/min→150°C (hold for 1min)→25°C/min→250°C;

C program temperature rise 70°C (hold for 2min)→40°C/min→150°C (hold for 1min)→25°C/min→250°C;

Enter the following data into the computer program: column phase β, initial concentration of the compound to be tested C _M00 , number of theoretical plates N, number of jumps n, six constant temperature values, virtual dead time τ, retention factor and peak shape parameters The parameters in the relationship with temperature, the initial temperature of the multi-stage temperature program, the temperature at the end of the first stage, the temperature at the end of the second stage, the heating rate of the first stage, the heating rate of the second stage, the temperature maintained at the initial temperature Time and the time maintained under the first-stage temperature program temperature termination temperature; run the computer program to obtain the predicted chromatographic peak shapes of heptanoic acid under the above-mentioned three temperature program temperature conditions respectively.

(4) Use the HP-5 column to obtain the corresponding chromatographic peak shapes of heptanoic acid under the above three programmed temperature conditions on the gas chromatograph, and compare them with the predicted peak shapes, as shown in Figures 5, 6, and 7 in turn.

It can be seen from the above respective comparison charts that the predicted peak shape has reached a high degree of agreement with the experimental peak shape, which confirms the accuracy of predicting the tongue-out chromatographic peak shape under different temperature programming conditions based on the nonlinear tray theory. the feasibility of the method.

In this embodiment, the prediction relative error %=(prediction result−experimental result)/experimental result×100. The results are shown in Table 2:

Table 2 The relative error between the predicted peak shape of heptanoic acid and the experimental peak shape under different programmed temperature conditions

Claims (1)

1. the method for a forecasting leading chromatographic peak shape under multi-order programmed temperature condition, the method is take non-linear plate theory as basis, for HP6890 gas chromatograph and nonpolar HP-5 chromatographic column, adopt the constant virtual dead time arbitrarily in forecasting process, it is characterized in that comprising following process:

1) setting of virtual dead time τ: chromatographic column design temperature variation range is 30-250 ℃, measures testing compound at T ₁=30 ℃, T ₂=50 ℃, T ₃=100 ℃, T ₄=150 ℃, T ₅=200 ℃ and T ₆Retention time t under=250 ℃ of six constant temperature _R1, t _R2, t _R3, t _R4, t _R5And t _R6, determine wherein minimum retention time value, all can be used as the virtual dead time with any one time value less than this minimum retention time value;

2) the retention time t under six constant temperature the calculating of Retention factor k under constant temperature: according to step 1) measuring _R1, t _R2, t _R3, t _R4, t _R5, t _R6The virtual dead time τ that has determined, employing formula 1, calculate retention factors corresponding under six constant temperature: k ₁, k ₂, k ₃, k ₄, k ₅And k ₆,

K=(t _R-τ)/τ formula 1

In formula 1: k is retention factors,

τ is the virtual dead time,

t _RRetention time for corresponding each temperature spot;

3) calculating of peak shape parameter (peak shape parameter, psp) under constant temperature:

Peak shape parameter psp is defined as the testing compound set up based on the non-linear plate theory coefficient in the funtcional relationship between the concentration in fixing and mobile phase on the same column plate, funtcional relationship as shown in the formula:

$C_S=\mathrm{psp}\&CenterDot;C_M^2+(k+1)\&CenterDot;C_M$ Formula 2

C _S+ C _M=C _nFormula 3

In formula 2: psp is the peak shape parameter value;

C _S, C _MBeing respectively testing compound fixes mutually and the concentration in mobile phase on the same column plate;

In formula 3: C _nFor testing compound concentration sum in fixing and mobile phase on the same column plate;

, with following computer program operation, calculate the peak shape parameter value under condition of different temperatures:

At first input known parameters in program: the initial concentration C of chromatogram column temperature T, testing compound under constant temperature _M00, Retention factor k, theoretical cam curve N and the number of skips l of testing compound under this temperature conditions; Recycling formula 2 and formula 3, input three different peak shape parameter values, and comparing calculation machine program is moved the peak shape and experiment peak shape that obtains respectively, draws the relation of variation tendency between peak shape parameter and peak shape size; Constantly adjust the size of the peak shape parameter value of input, until the peak shape under this constant temperature that obtains is while fitting like a glove with the experiment peak shape, this numerical value is just the actual peak shape parameter value of this testing compound under this temperature conditions;

Utilize the corresponding Retention factor k that jumps the i time _i, and the peak shape parameter value psp that sets,, according to formula 4 and formula 5, calculate respectively the concentration of testing compound and mobile phase mutually fixing the 1st, 2, in 3...n...N piece column plate:

$C_{\mathrm{Sni}}=\mathrm{psp}\&CenterDot;C_{\mathrm{Mni}}^2+(k+1)\&CenterDot;C_{\mathrm{Mni}}$ Formula 4

C _Sni+ C _Mni=C _niFormula 5

In formula 4: C _SniAnd C _MniBe respectively number of skips while being i, testing compound is the concentration in mutually fixing and mobile phase in n piece column plate;

In formula 5: C _niWhile for number of skips, being i, the total concentration of testing compound in n piece column plate, it is determined by formula 6:

C _ni=C _{S (n-1) i}+ C _{M (n-1) (i-1)}Formula 6

C _M00＝lμg/mL

In formula 6: C _M00For the initial concentration of testing compound,

C _{S (n-1) i}While for number of skips, being i, testing compound is in the fixing concentration in mutually of n-1 piece column plate,

C _{M (n-1) (i-1)}While for number of skips, being i-1, the concentration of testing compound in the mobile phase of n-1 piece column plate;

when testing compound jumps to last piece column plate, testing compound is still according to formula 4, formula 5 relations are distributed, when jumping next time, testing compound on last piece column plate in mobile phase flows out, record the concentration value that this flows out constantly, the testing compound that testing compound in fixing mutually and a upper column plate mobile phase are brought into re-starts distribution, so, until reach the number of skips of setting, guarantee that testing compound all flows out chromatographic column, obtain thus testing compound corresponding data point that flows out concentration value when jumping each time, and be the chromatographic peak profile that lolls of prediction by the figure that these data points form,

Set respectively T in above-mentioned computer program ₁, T ₂, T ₃, T ₄, T ₅And T ₆Six kinds of Temperature numerical, and obtain thus corresponding peak shape parameter value psp ₁, psp ₂, psp ₃, psp ₄, psp ₅And psp ₆

While 4) determining that testing compound once jumps arbitrarily in chromatographic column under the temperature programme condition, corresponding temperature T _i:

(1) determine each time △ τ that jumps and need with formula 6,

The formula 6 of △ τ=τ/(N-1)

In formula 6: τ is the virtual dead time, by step 1) definite,

N is the intrinsic theoretical cam curve of chromatographic column;

(2) testing compound jump i time in chromatographic column, t altogether takes time _i, calculated by formula 7,

t _i=ix △ τ formula 7

In formula 7, i is number of skips;

(3) in multistage temperature programme, calculate the T.T. t of multistage temperature programme:

T=t _h1+ t ₁+ t _h2+ t ₂Formula 8

In formula 8: t _h1For the retention time of initial temperature, empirical value is: 1-5min,

t _h2For the retention time of the final temperature of first stage temperature programme, empirical value is: 1-5min,

t ₁For the first stage temperature programme need the time,

t ₂For the subordinate phase temperature programme need the time,

t ₁And t ₂Calculated by formula 6 and formula 7 respectively:

t ₁=(T _m-T ₀)/r ₁Formula 9

t ₂=(T _f-T _m)/r ₂Formula 10

In formula 9: T _mFor the final temperature of first stage temperature programme,

T ₀For initial temperature,

r ₁Heating rate for the first stage temperature programme;

In formula 10: T _fFor the final temperature of subordinate phase temperature programme,

r ₂For the heating rate of subordinate phase temperature programme,

Wherein, r ₁And r ₂The experience span be 5-30 ℃/min;

While (4) determining that testing compound jumps for the i time, temperature T corresponding to chromatographic column this moment _i:

Work as t _i＜t _h1, column temperature T _i=T ₀,

Work as t _h1＜t _i＜(t _h1+ t ₁), column temperature T _i=r ₁* (t _i-t _h1)+T ₀,

As (t _h1+ t ₁)≤t _i≤ (t _h1+ t ₁+ t _h2), column temperature T _i=T _m,

As (t _h1+ t ₁+ t _h2)＜t _i＜t, column temperature T _i=r ₂* (t _it ₁-t _h1-t _h2)+T _m,

Work as t _i＞t, column temperature T _i=T _f

5) the determining of Retention factor k and peak shape parameter psp and temperature relation under multistage temperature programme condition:

(1) utilize six thermostat temperature value T ₁, T ₂, T ₃, T ₄, T ₅And T ₆The corresponding Retention factor k of trying to achieve and step 2) ₁, k ₂, k ₃, k ₄, k ₅And k ₆, by regretional analysis, the functional relation that obtains between Retention factor k and temperature T is formula 11:

Ink=aT ³+ bT ²+ cT+d formula 11

Wherein parameter a, b, c and d are definite value;

Calculate thus arbitrary temp point T in temperature programme _iCorresponding Retention factor k _i

(2) utilize six thermostat temperature value T ₁, T ₂, T ₃, T ₄, T ₅And T ₆And step 3) the corresponding peak shape parameter value psp that obtains in ₁, psp ₂, psp ₃, psp ₄, psp ₅And psp ₆, by regretional analysis, the functional relation that obtains between peak shape parameter psp and temperature T is formula 12:

Inpsp=a ' T ³+ b ' T ²+ c ' T+d ' formula 12

In formula 12, parameter a ', b ', c ' and d ' are definite value;

Calculate thus arbitrary temp point T in temperature programme _iCorresponding peak shape parameter psp;

6) acquisition of testing compound prediction peak shape under multistage temperature programme condition:

The following data of input in computer program: post is compared β, testing compound initial concentration C _M00, the initial temperature of theoretical cam curve N, number of skips n, six thermostat temperature values, virtual dead time τ retention factors and each parameter in peak shape parameter and temperature relation formula, multistage temperature programme, temperature that the first stage stops, temperature that subordinate phase stops, the heating rate of first stage, subordinate phase heating rate, initial temperature under two times of keeping under time of keeping and first stage temperature programme final temperature; By step 3) described in computer runs programs, obtain the chromatographic peak profile that lolls of the prediction of testing compound under this temperature programme condition.

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