Measuring and testing – Liquid analysis or analysis of the suspension of solids in a... – Content or effect of a constituent of a liquid mixture
Reexamination Certificate
1998-11-25
2002-03-26
Noland, Thomas P. (Department: 2856)
Measuring and testing
Liquid analysis or analysis of the suspension of solids in a...
Content or effect of a constituent of a liquid mixture
C210S198200, C428S304400
Reexamination Certificate
active
06360589
ABSTRACT:
TECHNICAL FIELD
The present invention relates to liquid chromatograph, and more particularly to a liquid chromatograph having a pre-focusing column used for condensing a specimen to be analyzed, and a method of analysis of a specimen including a condensation process.
PRIOR ART
Liquid chromatography is used extensively in the field of chemical analysis for separating and analyzing specimen quantitatively. Particularly, the liquid chromatography, which uses a semi-micro column having an inner diameter of 1-2 mm, is advantageous for providing a high sensitivity, high resolution and high precision analysis. Thus, intensive investigations are being made on the art of liquid chromatography.
In particular, there is an emphasis in the art of using a semi-micro column or a short general-purpose column (such as the one having an inner diameter of 4 mm and a length of 10 mm or 20 mm) for a pre-focusing column, not for a separation column, wherein the pre-focusing column is used for condensing the specimen.
The pre-focusing column is provided separately to the usual separation column in a liquid chromatograph, and is used prior to the separation of the specimen in the separation column. By using the pre-focusing column, impurities are removed from a sample solution and the specimen is condensed in the sample solution.
Inside the pre-focusing column, a column packing material corresponding to the specimen to be analyzed is provided, such that the sample solution is condensed by the foregoing column packing material.
Thus, the separation process in the separation column is made based on the condensed specimen solution, and the performance of separation in the separation column is improved significantly.
The use of a small-volume, semi-micro column for the purpose of pre-focusing column is particularly advantageous for improving the efficiency of condensation therein and hence for improving the performance of separation in the separation column.
On the other hand, a specimen to be analyzed generally contains a wide variety of impurities, and there can be a case in which the efficiency of condensation in the pre-focusing column is seriously deteriorated.
For example, the use of a liquid chromatograph for separating a mixture containing a blood serum causes a problem in that proteins contained in the blood serum with a large amount are adsorbed on the column packing material and the efficiency of condensation is impaired.
In order to avoid the problem of degrading of condensation in the column packing material and to secure a high resolution separation in the separation column, it has been necessary to remove the proteins from the specimen prior to the process of liquid chromatography by conducting a preparation process.
However, such a preparation process takes a time and a substantial work load. Further, such a preparation process tends to cause a degradation in the precision of analysis. In view of above, there are provided new column packing materials that allow a direct injection of specimen containing proteins into the liquid chromatograph without conducting the process of removing the proteins. The column packing material thereby achieves the condensation of the protein-containing specimen.
These improved column packing materials use porous glass or silica gel as a carrier, and a substance having a different property is provided on the surface of the pores. By using such an improved column packing material, the large protein molecules are refused to enter the fine pores. The large protein molecules merely pass through the column without being adsorbed on the hydrophilic surface (surface outside the pores), while small molecules of drugs are adsorbed by the hydrophobic inner surface (pore inner surface).
An example of such a column packing material is disclosed in the Japanese Laid-Open Patent Publication 60-56256. In the foregoing reference, the column packing material has a protein coating on a silica surface to which an octadecyl group is bonded. The protein used for the coating may be a bovine serum albumin, and the packing material is formed by adsorption and denaturation of the protein on the octadecyl-bonded silica surface.
On the other hand, such a protein-coated ODS silica packing material has a drawback in that the adsorbed denaturation protein tends to cause an elution when used for a long time. Further, such a packing material has a problem of durability or resolution in that it is difficult to provide a column of high separation efficiency.
In order to improve the foregoing problems, it is proposed, as described in the Japanese Laid-Open Patent Publications 61-65159 and 1-123145, to provide a method of producing a column packing material according to the steps of:
(1) introducing hydrophobic groups on the inner surface and outer surface of the porous support;
(2) selectively disconnecting the hydrophobic groups from the outer surface by using an enzyme, which is a macromolecules and cannot enter the pores of the silica support; and then
(3) introducing hydrophilic groups on the outer surface.
In more detail, the Japanese Laid-Open Patent Publication 61-65159 describes a process of: bonding oligopeptide on a porous silica as a starting material to which a glycerylpropyl group is introduced, via carbonyldiimidazole; and disconnecting the phenylalanine side chain on the outer surface by a hydrolysis using carboxypeptidaze A as a proteolytic enzyme. As a result of this, glycol-phenylalanylphenyl-alanine remains on the inner surface of the column packing material as a hydrophobic ligand, while the outer surface carries a hydrophilic glycyl-glycerylpropyl group thereon.
According to the process of the Japanese Laid-Open Patent Publication 1-123145, there is described a process of: introducing a hydrophobic group on the surface of a porous silica starting material, to which an aminopropyl group is introduced, via an amide bond, by causing a reaction with octanoyl chloride under existence of triethylamine; causing a hydrolysis in the acyl group on the surface by using polymyxin acylase; and making the amino group on the outer surface to be hydrophilic by conducting a reaction with glycydol.
When the foregoing column packing material disclosed in the Japanese Laid-Open Patent Publication 61-65159 or 1-123145 is used for the pre-focusing column of a liquid chromatograph, on the other hand, there arises a problem, associated with the fact that the disclosed packing materials are formed by using an enzyme reaction, in that the production of the column packing material is complicated and the obtained column packing material tends to show a variation in the performance.
Further, there has been a problem in the conventional liquid chromatograph, which uses a pre-focusing column of large volume of 2.0 ml or more, in that the specimen supplied thereto may be diluted rather than condensed. When this occurs, the precision of detection is deteriorated inevitably. This problem becomes particularly serious in the case in which the amount of the injected specimen is very small.
It should be noted that the foregoing packing materials have a common feature of removing a substance such as proteins. In order to remove proteins selectively, the surface of the foregoing conventional packing materials is controlled hydrophobic or hydrophilic. On the other hand, such a control does not enhance the separation effect of the specimen to be analyzed.
Thus, the specimen to be analyzed is still processed by the hydrophobic surface in the foregoing conventional packing material just similarly as before, and thus, the type of the specimens, particularly the range of the specimens subjected to the pre-focusing condensation process, is essentially the same as in the conventional case.
Thus, the specimen to be condensed is limited, in the foregoing prior art, to a non-polar or low-polar substance, and the prior art cannot achieve an effective pre-focusing condensation process for ionic substances contained in a biological specimen. This problem occurs when the prior art process is applied to the analysis of a biolo
Kanda Taketoshi
Ohkubo Aya
Ohtsu Yutaka
Yamaguchi Michihiro
Ladas & Parry
Noland Thomas P.
Politzer Jay L.
Shiseido Company Ltd.
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