JP4715221B2 - Porous silica continuous column - Google Patents

Description

 The present invention relates to a column for chromatography, particularly a column using a porous silica continuum.

 Separation technology using column packing, represented by high-performance liquid chromatography (HPLC), is a general technique for precise separation and quantification and preparative purification of natural and synthetic organic compounds, pharmaceuticals, and biological components. It is used in a wide range of fields. 70-80% of HPLC separation modes are reversed phase liquid chromatography, with solute partitioning mainly between the hydrophobic stationary phase and the hydrophilic mobile phase, and separation is achieved by the difference in hydrophobicity . In recent years, there has been a growing demand for separation and analysis of highly ionic and water-soluble samples such as biologically derived components, and examples of use of ion exchange liquid chromatography and hydrophilic interaction liquid chromatography are also increasing.

 The advantages of this reversed-phase liquid chromatography are 1) wide range of target compounds, 2) 2 to 3 types of solvents including water, and 3) It is easy to obtain reproducibility in the results, and 4) it is not necessary to expose the sample to high temperature. As column fillers for reversed-phase liquid chromatography, chemically bonded silica gel fillers and cross-linked organic polymer fillers are widely used. Chemically bonded silica gel fillers have high column efficiency but have few types of chemical modification methods. In addition, it has characteristics such as instability under acid and base conditions. On the other hand, crosslinked organic polymer fillers have relatively low column efficiency, but have many types of chemical modification methods and are relatively stable against acids and bases. It has the characteristic of being.

 That is, there are many micropores (pores of 1 to 2 nm or less) in the crosslinked organic polymer filler, and a solute that matches the size and shape of this part enters, resulting in slow diffusion and low column efficiency. Regarding chemical modification, various chemical modifications of the surface are possible, and high stability is also obtained chemically (under acidic and basic conditions).

 On the other hand, in the chemically bonded silica gel filler, high column performance can be obtained because the solute diffusion in the mesopores (2-50 nm pores) of the silica gel particles is easy. However, when chemical modification is performed, there is a limitation on the functional group that reacts with the silanol group, so that the chemical modification method is limited, secondary retention effect due to residual silanol groups and metal impurities occurs, high Problems such as dissolution of silica gel at pH (> 8), or cleavage of silicon-oxygen bonds at low pH (<2).

 In general, it is not easy to optimize packing conditions when forming a column for packing materials other than reversed-phase silica gel packing, and packing with different separation modes such as reversed-phase type, fluorine-containing type, and ion-exchange type. It is difficult to obtain a high-performance column by packing the agent stepwise or thoroughly mixed.

A silica continuous column having a three-dimensional network structure silica skeleton has a fundamentally different column forming process from a particle packed column and does not require a packing operation. Due to its high permeability, the liquid feeding pressure was low, and separation was possible under ultra-high speed conditions that could not be operated using a particle packed column. Moreover, the improvement in separation performance due to the thin silica skeleton is remarkably improved, and it is possible to provide a separation performance that is an order of magnitude higher than that of the particle-packed type (Patent Document 1).
In the silica continuous column, the amount of the silica skeleton per column volume is only about 10 to 20%, and the introduction amount of the identification substituent is inevitably reduced. Compared with a reversed-phase column, the retention coefficient of the sample can be obtained only about one third of that of the particle-packed type, and a small amount of the sample is also a drawback.
Furthermore, it is difficult for the interaction to cancel each other out by mixing, so that a desired plurality of separation modes can be added simultaneously.

JP-A-6-265534

One of the research needs in the post-genome era is that there is an increasing need for comprehensive separation and analysis of highly complex multi-component samples such as proteins, peptides, lipids, sugars, and metabolites. . However, the separation performance of ion-packed, hydrophilic interaction, and size-excluded particle-packed columns is clearly lower than that of reversed-phase columns and cannot achieve 10,000 theoretical plates. As a result, there is a lack of performance for exhaustive separation analysis of complex components.
The present invention has been made in view of the above-described problems of the prior art, and an object thereof is to create a column that provides various separation modes while maintaining the high separation performance of the silica continuous column support. It is also aimed to create a column that achieves a larger sample retention capacity than conventional silica continuum columns and that the silica support surface does not contact the sample.

In order to achieve the above object, the present inventors have conducted intensive studies. As a result, the porous silica continuum is formed into a film, and the surface of the silica support and the sample are contacted by alternately stacking films having different surface modifications. As a result, the present invention has been completed by making it possible to provide a variety of separation modes by increasing the retention capacity.
That is, the present invention provides a porous silica continuum column in which membranes having different surface modifications are alternately stacked on the porous silica continuum. In the present invention, the term “film” is a concept including a disk or a plate. In addition, unmodified films without surface modification may be alternately stacked, or unmodified films and modified films may be stacked alternately.
The porous silica continuum is prepared by causing a sol-gel transition accompanied by phase separation in a reaction solution containing silica as a main component.
The porous silica continuum is an inorganic porous continuum in which macropores having a diameter of 100 nm to 10000 nm and a skeleton have a co-continuous structure, and the skeleton has a mesopore having a diameter of 2 nm to 50 nm. However, the mesopores may be reduced by baking after the sol-gel transition.
If the porous continuous silica column of the present invention is used, for example, it is optimal for high-speed separation of glycohemoglobin. In this case, it is preferable to use a modified film of 3-sulfopropyl potassium methacrylate and a modified film of octadecyldimethylchlorosilane alternately in two to five layers, preferably three layers.

The porous silica continuous body in the present invention can be obtained by causing a reaction solution containing silica as a main component to undergo a sol-gel transition accompanied by phase separation. Examples of the precursor of the network component that causes gel formation used in the sol-gel reaction include metal alkoxides, complexes, metal salts, organically modified metal alkoxides, organic cross-linked metal alkoxides, and partial hydrolysis products and partial polymerization products thereof. Can be used. Sol-gel transition by changing the pH of water glass and other silicate aqueous solutions can be used as well.
More specifically, the means for achieving the above-mentioned object is to generate a water-soluble polymer and a compound that thermally decomposes in an acidic aqueous solution, add a metal compound having a hydrolyzable functional group, and perform a hydrolysis reaction. After the product has solidified, the wet gel is preferably heated to thermally decompose the low molecular weight compound previously dissolved at the time of gel preparation, and then dried and heated.

   Here, the water-soluble polymer is theoretically a water-soluble organic polymer that can be formed into an aqueous solution having an appropriate concentration, and in a reaction system including an alcohol generated by a metal compound having a hydrolyzable functional group. The polymer metal salt is a sodium salt or potassium salt of polystyrene sulfonic acid, which is a polymer metal salt, polyacrylic acid which is a polymer acid and dissociates to form a polyanion, Polyallylamine and polyethyleneimine that are molecular bases that generate polycations in aqueous solution, or neutral polymers that are polyethylene oxide having an ether bond in the main chain and polyvinylpyrrolidone having a carbonyl group in the side chain are suitable. . In addition, formamide, polyhydric alcohol, or surfactant may be used in place of the organic polymer. In this case, glycerin is optimal as the polyhydric alcohol, and polyoxyethylene alkyl ethers are optimal as the surfactant.

 As the metal compound having a hydrolyzable functional group, a metal alkoxide or an oligomer thereof can be used, and those having a small number of carbon atoms such as a methoxy group, an ethoxy group, and a propoxy group are preferable. Further, as the metal, an oxide metal finally formed, for example, Si, Ti, Zr, or Al is used. The metal may be one type or two or more types. On the other hand, any oligomer can be used as long as it can be uniformly dissolved and dispersed in alcohol. Specifically, it can be used up to about 10-mer.

 Moreover, as acidic aqueous solution, the thing of 0.001 mol concentration or more of mineral acids, such as hydrochloric acid and nitric acid, or 0.01 mol concentration or more of organic acids, such as an acetic acid and a formic acid, is preferable normally.

  The phase separation and gelation can be achieved by storing the solution at room temperature of 40 to 80 ° C. for 0.5 to 24 hours. Phase separation / gelation is a process in which an initially transparent solution becomes cloudy, causing phase separation between a silica phase and an aqueous phase and finally gelling. By this phase separation / gelation, the water-soluble polymers are in a dispersed state and their precipitation does not substantially occur.

Specific examples of the thermally decomposable compound that coexists in advance include urea or hexamethylenetetramine, formamide, N-methylformamide, N, N-dimethylformamide, acetamide, N-methylacetamide, N, N-dimethylacetamide and the like. However, since the pH value of the solvent after heating is an important condition, there is no particular limitation as long as it is a compound that makes the solvent basic after thermal decomposition.
Although the thermally decomposable compound to coexist depends on the kind of the compound, for example, in the case of urea, it is 0.05 to 0.8 g, preferably 0.1 to 0.7 g, with respect to 10 g of the reaction solution. The heating temperature is preferably 40 to 200 ° C. in the case of urea, for example, and the pH value of the solvent after heating is preferably 6.0 to 12.0.
Moreover, what produces the compound with the property to melt | dissolve a silica like hydrofluoric acid by thermal decomposition can be utilized similarly.

  The porous silica continuum may have mesopores or be burned to disappear. The firing temperature is, for example, 600 ° C to 1200 ° C, preferably 950 ° C to 1050 ° C. In particular, the specific surface area, mesopore pore diameter, and pore volume greatly decrease from 950 ° C. to 1050 ° C., and the mechanical strength increases accordingly.

The surface-modified film is produced, for example, by introducing an ion exchange group through a hydrophobic modifier or a spacer. As the hydrophobic modifier, for example, alkyldimethylchlorosilane such as octadecyldimethylchlorosilane, alkylmethyldichlorosilane, or alkyltrichlorosilane can be used, and examples of the spacer include methacryloylpropyltrimethoxysilane (triethoxy). As the ion exchange group, methacrylic acid 3-sulfopropyl potassium salt, 3-aminopropyltriethoxysilane, (meth) acrylic acid, 4- (2-) vinylpyridine, 4-vinylphenylsulfonic acid Etc. can be used.
In these modifications, 1 to 10 equivalents, preferably 5 equivalents, of silanol is added to the porous silica continuum, and the reaction is carried out at a temperature of 50 ° C to 120 ° C, preferably 60 ° C to 110 ° C. For example, ODS (octadecyldimethylchlorosilane) and pyridine were added in an amount equivalent to 5 equivalents of the amount of silanol groups present on the surface of the porous silica continuous film, and reacted for 24 hours under toluene reflux conditions. In the preparation of the cation exchange membrane (MASK membrane), the porous silica continuous membrane is coated with MOP (3-methacryloxypropyltrimethoxysilane) and 2,6-lutidine as spacers in the toluene solvent. 5 equivalents of silanol groups present in each were added, reacted for 24 hours under toluene reflux conditions, and then washed with toluene and methanol. After the MOP as a spacer is modified on the surface of the porous silica continuous membrane, MASK (3-sulfopropyl methacrylate) is equivalent to 5 equivalents of silanol groups present on the silica membrane surface and methanol is used for 24 hours using BPO as the initiator. After reacting under reflux conditions, it was washed with water and methanol.
In the preparation of the anion exchange membrane, APS (3-aminopropyltriethoxysilane) and pyridine were added to the porous silica continuous membrane at 5 equivalents each of the silanol groups present on the surface of the porous silica continuous membrane, and the toluene reflux conditions were maintained for 24 hours. The reaction was carried out below, followed by washing with toluene and ethanol.
The surface modification film may be a block body or a laminate.
The type of the modified membrane can be selected according to the separated sample. For example, when separating glycated hemoglobin, a plurality of layers, preferably three or more layers, of a surface-modified membrane having an ion exchange group and a reversed-phase modified membrane are alternately laminated. In addition to this, for example, when separating proteins, amino acids and the like, a plurality of different ion exchange group-modified membranes are laminated.

Preparation of Porous Silica Monolith 50 ml of 0.01 N acetic acid aqueous solution was added to 4.2 g of polyethylene glycol (PEG) having a molecular weight of 10,000 and dissolved by stirring. After complete dissolution, the mixture was cooled on ice, and 24 ml of tetramethoxysilane was added and stirring was continued. The solution was transferred to a square container, a Teflon ring was inserted, sealed, and left in a constant temperature bath (water bath) for 40 days. The obtained silica bulk was immersed in an aqueous solution of ethanol / water = 1/1 (v / v) and left for 1 day. After immersing this bulk in 0.5 M urea aqueous solution and heat-treating it using a Teflon decomposition bottle,
The porous silica monolith was prepared by transferring it to a 40 ° C oven and leaving it for 2 days, and then transferring it to a 60 ° C oven and leaving it for 1 day, followed by firing using an electric furnace.
Preparation of porous silica continuous membrane A porous silica monolith is processed into a cylindrical shape (membrane shape) with a diameter of 12 mm and a thickness of 3.0 mm, and a porous shrinkable tube (PTFE) is wound around this to form a continuous porous silica monolith. A body membrane was prepared.

Modification of octadecyldimethylchlorosilane (ODS) and hexamethyldisilazane (HMDS) on porous silica continuous membrane Azeotropically with ethanol and toluene to remove water from the reaction system, and then in toluene solvent Add octadecyldimethylchlorosilane (ODS) for 5 equivalents of silanol groups present on the surface of the porous silica continuous membrane, reflux for 24 hours, and then toluene, tetrahydrofuran, ethanol / water = 1/1 (v / v) , Washed in order of ethanol, and dried under reduced pressure. A 20% toluene solution of hexamethyldisilazane (HMDS) was refluxed for 17 hours on a porous silica continuous membrane converted to ODS, and washed with toluene, tetrahydrofuran, and ethanol in this order.

3-methacryloxypropyltrimethoxysilane (MOP) and 3-sulfopropyl potassium methacrylate (MASK) modified porous silica continuous film on porous silica continuous film as spacer in toluene solvent Roxypropyltrimethoxysilane (MOP) and 2,6-lutidine were added in increments of 5 equivalents of silanol groups present on the surface of the porous silica continuous membrane, refluxed for 24 hours, and then washed with methanol.
After the spacer MOP is modified on the surface of the porous silica continuous membrane, 5-sulfopropyl potassium methacrylate is equivalent to 5 equivalents of silanol groups present on the silica membrane surface, and benzoyl peroxide (BPO) is used as the initiator. The mixture was refluxed in a methanol solvent for 24 hours and then washed with water and methanol.

Modification of 3-aminopropyltriethoxysilane (APS) to porous silica continuous film Porous silica continuous film with 3-aminopropyltriethoxysilane (APS) and pyridine in toluene solvent to porous silica continuous film 5 equivalents of silanol groups present on the surface were added and refluxed for 24 hours, followed by washing with toluene and ethanol.

Characteristics of porous silica continuous film
1) Pore observation by BET method Measurement of BET specific surface area (m2 / g), mesopore pore diameter (nm), pore volume (cm3 / g) using a surface area measuring device (Gemini 2375 [V 5.0]) Went.
The BET specific surface area (m2 / g), mesopore pore diameter (nm), and pore volume (cm3 / g) were measured by changing the firing temperature. The specific surface area and pore volume tended to decrease when the firing temperature was increased from 600 ℃ to 1050 ℃. In particular, it was confirmed that the specific surface area, mesopore pore diameter, and pore volume decreased greatly from 950 ° C to 1050 ° C, and the mechanical strength increased accordingly. This is because the melting point of silica exists near 1000 ° C, and firing near this temperature causes extreme shrinkage of the silica monolith skeleton, resulting in a significant decrease in specific surface area, mesopore pore diameter, and pore volume. Seems to have brought.
Since the membrane column is a hand-clamped column, a mechanical load is applied to the porous silica continuous membrane, which is the separation medium, to some extent. It was decided to use a porous silica continuous membrane.

Analysis of pore characteristics by HPLC measurement Porosity calculation When the porous silica continuous membrane is inserted into the column and when it is not, the respective t0 (elution of solute without retention) Time) was measured, and the amount of voids in the porous silica continuous membrane was calculated from the flow rate at that time. From this value and the apparent total volume of the porous silica continuous film, the porosity (porosity) of the porous silica continuous film was calculated (porosity p = 0.651).

HbA1c analysis
1) Comparison of HbA1c analysis by MASK membrane and MASK & ODS membrane The abbreviations were given when the porous silica continuous membrane was inserted into the membrane column in order from the upstream side in the flow direction as follows. If it is necessary in the future, it shall be described according to this law.
ODS membrane alone ⇒ “O”
MASK membrane alone ⇒ “M”
In the order of MASK film → ODS film ⇒ “MO”
In the order of MASK film → ODS film → MASK film → ODS film → MASK film → ODS film ⇒ “MOMOMO”
MASK film → MASK film → MASK film → ODS film → ODS film → ODS film in this order ⇒ “MMMOOO”
In the measurement of HbA1c by HPLC, the Hb component is separated into HbA0, HbA1a + b, and HbA1c fractions by the principle of ion exchange chromatography, and the HbF, L-HbA1c, St. It is known to isolate -HbA1c.
We investigated the extent to which these two actions contributed to the separation of the “cation exchange interaction” and “hydrophobic interaction” required for the separation of HbA1c.
Evaluation was performed using SP-NPR (TSK-gel), a commercially available cation exchange column, as a reference column for elution of Hb samples (Fig. 1).
First, in order to confirm the contribution of the cation exchange interaction, an analysis by HPLC using “M” and “MO” was attempted (FIG. 2).

HPLC conditions
Column: Membrane column (“M” or “MO”)
Flow rate: 0.4 ml / min
Detection: 415 nm
Temperature: 25 ° C
Eluent:
(A); 20 mM MES, 20 mM HEPES, 0.01% NaN3 (pH 5.20)
(B); 20 mM MES, 20 mM HEPES, 400 mM NaCl, 0.01% NaN3 (pH7.00)
Injection volume: 3 μl
Gradient table:
Time (min) 0 10 16 17 35 36 38 39 50
B (%) 18 19 23 26 28 80 80 18 Stop run

From this result, it was confirmed that “M” can separate HbA1c to some extent.
However, as can be seen from Fig. 2, there was a phenomenon in which the HbA1c peak was absorbed by the HbA0 peak eluting later as the number of injections increased. In addition, the “MO” resolution is clearly superior to the “M” resolution compared to HbA1c, and this comparison indicates that the need for an ODS membrane is greater.
As for aging, “M” requires only 1 to 2 times, but “MO” requires about 10 sample injections, and “M” is one of the problems of HbA1c analysis. Was found to be advantageous. This suggests that aging is associated with hydrophobic interaction by ODS membrane.

2) Comparison of HbA1c analysis with ODS and MASK & ODS membranes Next, in order to confirm the contribution of hydrophobic interaction, an analysis by HPLC using “O” and “MO” was attempted. The analysis conditions are the same as [0026].
From the results of this experiment (Fig. 3), it was confirmed that "O" cannot separate HbA1c at all.
When compared with “M”, the separation performance was clearly different, confirming that the cation exchange interaction contributed greatly to the separation of HbA1c from other fractions. However, even when measured by “MO”, the HbA1c peak was a single peak, and no separation tendency of L-HbA1c and St-HbA1c was observed.
As for aging, the comparison between “O” and “MO” showed that the number of aging hardly changed by inserting MASK film. However, when the evaluation by “O” was performed from this experimental result, the phenomenon that the peak was separated into two after the aging was completed. The possible cause is that the blood protein adsorbed on the ODS membrane at the aging stage interacted with the hemoglobin sample.
In the “MO” evaluation, the peak of HbA1c tends to shift forward after aging is completed. This may also suggest an interaction with blood proteins adsorbed on the ODS membrane.
In this time, the definition of aging was defined as the time when the fluctuation of the elution peak area ceased, but if the condition until the fluctuation (movement) of the peak ceases to be considered as conditioning, aging and another action seem to contribute to conditioning. It seems to be. Although it is not certain about this other action, it was thought by this experiment that it might be caused by the adsorption of blood proteins. Further investigation is necessary in this regard.
Another observed phenomenon was that the peak shape changed significantly immediately before and immediately after the interruption by interrupting the evaluation once (several hours) and restarting the feeding. This change was a phenomenon in which the entire peak shape immediately after resumption became similar to the peak shape before injection several times from the one immediately before interruption. This can also be attributed to the action of protein adsorption in the blood.
These inferences are due to the fact that only the ODS membrane is colored red after evaluation, and the adsorption of Hb protein can be predicted from the blood protein, especially its color.
From these facts, it can be inferred that at least Hb protein is adsorbed to the hydrophobic part (mainly ODS membrane) in the blood protein, and this adsorption causes the hydrophobic interaction between the hemoglobin sample and the ODS membrane. It was suggested that there was a possibility of weak adsorption.

3) Comparison of “MO1” and “MO2” using ODS films with different lengths (“O1” and “O2”) From the results so far, it is almost certain that the ODS film is involved in aging. It is. Therefore, using ODS membranes with total length (thickness) of 3.5 mm (“O1”) and 7.7 mm (“O2”), how much hydrophobic interaction by ODS membrane contributes to aging. This was confirmed by evaluating “MO1” and “MO2” (FIG. 4). The HPLC conditions used for the evaluation are the same as [0026].
Previous studies have shown that almost no aging is observed when non-porous, nonporous fillers are used, and hemoglobin has a spherical size with a molecular size of approximately 6.4 x 5.5 x 5.0 nm. Since it is a molecule, it has been argued that the presence of pores near 5.0 nm causes the “aging” phenomenon to occur when Hb molecules are trapped in the pores, but the exact cause of the adsorption phenomenon is still determined. Was not.
In this experiment, we used a 3.5mm (“O1”) film and a 7.7mm (“O2”) film as the ODS film combined with the previously inserted MASK film. Comparing the number of times, it was found that “MO2” using “O2”, which is about twice as thick as “O1”, has about twice the aging frequency.
In the porous silica continuous membrane used this time, there are a minute amount of micropores called mesopores around 4.0 nm. However, since the mesopores themselves are very small and the pore size distribution is concentrated around 4.0 nm, the aging phenomenon is caused only by the entire Hb molecules getting stuck in the pores. Very Hard to think. This suggests that only a part of the Hb molecule fits into the pores of the porous silica continuous film, and that the process showing hydrophobic interaction may contribute to aging.

4) Faster HbA1c analysis
Acceleration of HbA1c analysis under high flow rate conditions Because “MO” has enabled HbA1c analysis to some extent, we attempted to increase the speed of HbA1c analysis, which is the ultimate goal.
As the first process, the speed was increased by reducing the flow rate to 9.99 ml / min (25 times that of the conventional method) and the time program to 1/25 each compared to the analysis conditions used up to the previous time.

HPLC conditions
Column: Membrane column (“MO”)
Flow rate: 9.99 ml / min
Detection: 415 nm
Temperature: 25 ° C
Eluent:
(A); 20 mM MES, 20 mM HEPES, 0.01% NaN3 (pH 5.20)
(B); 20 mM MES, 20 mM HEPES, 400 mM NaCl, 0.01% NaN3 (pH7.00)
Injection volume: 3 μl
Gradient table:
Time (min) 0 0.40 0.64 0.68 1.40 1.44 1.52 1.56 2.00
B (%) 18 19 23 26 28 80 80 18 Stop run

In this study, the analysis was simply performed under high flow rate conditions in order to achieve high speed.
This method certainly enables high-speed analysis and shortens the analysis time, but performing a gradient analysis of a complex time program at a high flow rate places a burden on the device, and the reproducibility of the data obtained is also high. It was low and it was not possible to identify which peak was HbA1c.

Optimization of analysis conditions for HbA1c (1)
In the above measurement, the flow velocity was so fast that the system was not stable and reproducible data could not be obtained. Therefore, in order to overcome this situation, an attempt was made to improve the gradient conditions and mobile phase composition. . First, the flow rate was 4.0 ml / min (10 times that of the conventional method) and the time program was set to 1/10 each in the range where the device could stably mix the gradient (Fig. 5).

HPLC conditions
Column: Membrane column (“MO”)
Flow rate: 4.00 ml / min
Detection: 415 nm
Temperature: 25 ° C
Eluent:
(A); 20 mM MES, 20 mM HEPES, 0.01% NaN3 (pH 5.20)
(B); 20 mM MES, 20 mM HEPES, 400 mM NaCl, 0.01% NaN3 (pH7.00)
Samples: Normal level sample, High level St-HbA1c
sample (abnormal sample)
Injection volume: 3 μl
Gradient table:
Time (min) 0 1.0 1.6 1.7 3.5 3.6 3.8 3.9 5.0
B (%) 18 19 23 26 28 80 80 18 Stop run

When measuring with this method, reproducible data was obtained because the instrument was able to mix gradients stably. In addition, it was possible to analyze only a very long time, which was one sample in 60 minutes until now, but this trial has achieved a certain speedup of one sample in 5.0 minutes.
The ratio of HbA1c was calculated from the chromatogram obtained this time (Table 1).
From this table, the percentage of HbA1c in the normal sample is 7.5%, the percentage of HbA1c in the abnormal sample is 12.2%, and the normal sample is classified as “possible” when compared with the percentage of normal adult HbA1c (Table 2). Although it seems to show a slightly high price, the result was reasonable to some extent.
Table 1. Percentage of HbA1c Normal sample 7.5%
Abnormal sample 12.2%
Table 2 Normal adult HbA1c ratio (reference values)
Evaluation of control Excellent Yes No
HbA1c (%) Less than 5.8 5.8 to 6.5 6.6 to 7.9 8.0 or more

In addition, from Fig. 5, the separation tendency (L-HbA1c and St-HbA1c) was observed in the HbA1c peak. This tendency was not observed when one sample was handled in 60 minutes, so the effect of shortening the analysis time may contribute to the improvement of the resolution.
However, at the forefront of HbA1c analysis, high-speed analysis of one sample is performed in 1.5 minutes, and it can be said that the true result of “acceleration” has not been achieved.
Based on the above, in the two methods shown so far, the limit of flow velocity becomes the limit of shortening the analysis time, and even if reproducible data is obtained, it will be one sample in 2.0 minutes at the shortest. Therefore, since it is impossible to aim for higher speeds, it was necessary to further improve the analysis conditions.

Optimization of HbA1c analysis conditions (2)
In the measurement so far, it is possible to increase the flow rate to a certain degree by increasing the flow rate, but performing a gradient analysis at a flow rate above a certain level (5.0 ml / min or more) places a heavy burden on the device and is reproducible. Since it was confirmed that some data could not be obtained, we examined whether it is possible to measure in isocratic mode with less burden even under high flow rate conditions.
New eluent with eluent A / eluent B = 72/28 (v / v) from the elution time of the HbA1c peak in the chromatogram obtained under the previous conditions and the gradient conditions (mobile phase composition) A new eluent Y having X and eluent A / eluent B = 20/80 (v / v) was prepared and analyzed using these two eluents. In this analysis, a porous silica continuous membrane with an inner diameter of 12.0 mm and a thickness of 2.0 mm was used. By processing to this size, it was possible to insert up to 6 porous silica continuous membranes (total length: 12.0 mm), which was considered to be advantageous for showing the usefulness of overlaying. .
First, using "MMMOOO", we tried to analyze HbA1c under new analysis conditions (Fig. 6).

HPLC conditions
Column: Membrane column (“MMMOOO”)
Flow rate: 2.00 ml / min
Detection: 415 nm
Temperature: 25 ° C
Mobile phase: Eluent X; Eluent A / Eluent B = 72/28 (v / v)
Eluent Y; Eluent A / Eluent B = 20/80 (v / v)
Method: TO-08
Samples: Normal level sample, High level St-HbA1c
sample (abnormal sample)
Injection volume: 3 μl
Gradient table:
Time (min) 0 0.30 0.31 0.70 0.71 1.00
Y (%) 0 0 100 100 0 Stop run

In order to compare the separation performance by overlapping the membranes, we tried to analyze HbA1c in “MOMOMO” using the same analysis conditions as those used in “MMMOOO” (Fig. 6).
As is clear from FIG. 6, there was a difference in separation performance between “MMMOOO” and “MOMOMO”. The inserted separation media have three MASK membranes and three ODS membranes (all membrane thicknesses are 2.0 mm) and the capacity is the same, but there is a large difference in performance. This suggests that the stacking order greatly contributes to the separation performance.
In the present invention, “MOMOMO”, which showed very good separation performance, was used to change the time program of flow velocity and gradient conditions to the limit, and tried to maximize the speed of HbA1c analysis at this stage (Fig. 7). ).
HPLC conditions
Column: Membrane column (“MMMOOO”)
Flow rate: 3.00 ml / min (TO-10), 4.00 ml / min (TO-09)
Detection: 415 nm
Temperature: 25 ° C
Mobile phase: Eluent X; Eluent A / Eluent B = 72/28 (v / v)
Eluent Y; Eluent A / Eluent B = 20/80 (v / v)
Method: TO-09, TO-10
Samples: Normal level sample, High level St-HbA1c
sample (abnormal sample)
Injection volume: 3 μl
Gradient table:
TO-10 Time (min) 0 0.20 0.21 0.46 0.47 0.66
Y (%) 0 0 100 100 0 Stop run
TO-09 Time (min) 0 0.15 0.16 0.35 0.36 0.50
Y (%) 0 0 100 100 0 Stop run

Table 3 Ratio of HbA1c at each flow rate when speeding up
2.0 ml / min 3.0 ml / min 4.0 ml / min
Normal sample 6.3% 6.0% 4.5%
Abnormal sample 10.3% 10.5% 8.3%

In the chromatogram in Fig. 7, high-speed separation from HbA0 to HbA1c was possible in the analysis up to a flow rate of 3.0 ml / min, and the separation tendency of St-HbA1c and L-HbA1c was also confirmed. In addition, Table 3 shows that the HbA1c ratio was fairly close to the measurement up to a flow rate of 3.0 ml / min, and it was possible to analyze HbA1c as fast as 1 sample per 1.8 minutes.
In the analysis at a flow rate of 4.0 ml / min, a large peak was detected before the HbA1c peak. This part may contain a part of HbA1c that could not participate in the adsorption, which may hinder the accurate value of HbA1c. Actually, Table 3 also shows that the value of HbA1c calculated under the condition of 4.0 ml / min is slightly smaller, suggesting that this analysis is limited to 3.0 ml / min. .
By analyzing under such conditions, it was possible to analyze a maximum of 1 HbA1c value, which could previously be analyzed only once every 60 minutes, and using a special column called a membrane column. It can be said that the acceleration of HbA1c analysis was achieved.
This is comparable to the required analysis time (one sample per 1.5 minutes) of a fully automated glycohemoglobin measuring device (Arkray, Inc.) that is currently on the market, and future possibilities are expected.

・ Membrane columns capable of applying a porous silica continuous membrane were fabricated and introduced into the HPLC system. As a result, it was confirmed that their performance was demonstrated in both reversed-phase chromatography and ion-exchange chromatography modes.
・ Evaluate HPLC characteristics quickly and easily by processing any polymer that is larger than a certain size but difficult to process as a general column (this time divinylbenzene polymer monolith) into a film. As a result, the characteristics of the membrane column that “any polymer can be processed into the column” were confirmed.
・ Additional improvements in additive performance were confirmed for both porous silica continuum films (ODS film and ODS film) with the same properties and porous silica continuum films (ODS film and MASK film) with different properties. This confirms the characteristics of the membrane column, which is “improvement of performance by overlapping separation media”.
• It was confirmed that HbA1c can be separated by overlaying MASK and ODS membranes. Furthermore, multiple MASK and ODS films processed to a thickness of 2.0 mm were alternately stacked, and the analysis conditions were improved, enabling high-speed analysis of HbA1c (analysis of one specimen in approximately 100 seconds). . This is almost the same as the required analysis time of a fully automated glycohemoglobin measuring device currently on the market, and it can be said that considerable speedup has been achieved. In addition, separation of St-HbA1c and L-HbA1c was achieved to some extent.

Example of HbAlc analysis by SP-NPR Changes in dissolution behavior in “M” and “MO” Change of dissolution behavior in “O” and “MO” Change of dissolution behavior in “MO1” and “MO2” Optimization of analysis conditions Optimization of analysis conditions (“MMMOOO” and “MOMOMO”) Optimum conditions for HbAlc analysis

Claims (4)

A porous silica continuum column in which membranes having different surface modifications are alternately stacked on a porous silica continuum, wherein the mesopore of the porous silica continuum phase-separates a reaction solution mainly composed of silica. A porous silica continuum column which is fired to cause a sol-gel transition and then fired to 4.0 nm or less . The surface-modified film is selected from octadecyldimethylchlorosilane, hexamethyldisilazane, 3-methacryloxypropyltrimethoxysilane, 3-sulfopropyl potassium methacrylate, and 3-aminopropyltriethoxysilane. Porous silica continuum column. A method for using a porous continuous column, wherein glycohemoglobin is separated using the porous continuous silica column according to claim 2 . The method of using a porous continuous column according to claim 3 , wherein three modified layers of 3-sulfopropyl potassium methacrylic acid salt and three octadecyldimethylchlorosilane modified membranes are alternately stacked.

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