Abstract:
Disclosed is a method for separating a plurality of different chemicals in a sample. The method uses a hexyl-bonded stationary phase composition in reversed-phase liquid chromatography. The composition includes a polymer-based chromatographic support composed of chemically stable and porous particles having a surface. The surface of said particles having hexyl-phenyl bonded thereto. A chromatography column is packed with this composition and a sample including a plurality of different chemicals is injected. Liquid is applied to force the sample through the column, whereby said different chemicals come out of the column at different times.

Description:
RELATED APPLICATIONS  
       [0001]    This application is a continuation of Application No. 08/909,267, filed Aug. 11, 1997, now allowed. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    The present invention relates generally to reversed-phase liquid chromatography. More specifically, the present invention relates to a chemical composition used as a stationary phase in the reversed-phase liquid chromatography, a method for preparing the composition, a column used in the liquid chromatography, and a method for performing the liquid chromatography.  
           [0004]    1. Description of related art  
           [0005]    Reversed-phase liquid chromatography is the most popular separation method in liquid chromatography. More than 50 percent of all the applications in liquid chromatography are performed in reversed-phase mode. This popularity stems from a number of factors such as ease of use, rapid analysis time, and commercial availability of a large number of reversed-phase columns. Besides, the theory of reversed-phase liquid chromatography is also very well developed.  
           [0006]    Early, many of the reversed-phase systems were developed using silica gel as the stationary phase matrix. However, conventional silica gel based packing materials have several disadvantages.  
           [0007]    One disadvantage relates to the presence of acidic/active silanol groups. The presence of unreacted silanol groups lead to the adsorption of basic chemicals on the column, resulting sometimes in tailing peaks or complete disappearance. To alleviate this, the use of masking agents like alkylamine or the use of low pH mobile phases which would protonate the silanols became common practice.  
           [0008]    Another disadvantage relates to pH stability. Conventional silica gel based packing materials have limited range of pH stability (2.5 to 7.5). At low pH, the silica-carbon bonds break down leading to the erosion of the bonded phase. At high pH, on the other hand, the silica gel itself dissolves, resulting in a loss of bonded phases. In both these instances, there is a change in the chromatographic profile. Recently, silica-based alkyl bonded phases which are stable to extended range of pH have been synthesized. Popular alkyl bonded phases are pentyl, octyl, and octadecyl bonded silica gels (respectively C5, C8, and C18) which are stable in the range of pH 1.50 to 10.0.  
           [0009]    The separation power of liquid chromatography, on the other hand, can be attributed to the unlimited selectability of mobile phase conditions. The optimal utilization of the power of liquid chromatography requires development of new stationary phases. In this regard, another popular phase in reversed-phase liquid chromatography which has different and unique selectivities compared to the alkyl (C5, C8, and C18) bonded phases is phenyl phase (C n Ph). Certain phenyl bonded phases have been developed and their phenyl groups are generally bound with silica gel via zero to four methylene groups; i.e., n=0, 1, 2, 3, or 4 in (C n Ph). However, these phases have limited pH stability and show poor retention capacity compared with the octyl (C8) and octadecyl (C18) bonded phases.  
           [0010]    As a result, there is a need for a new phenyl bonded phase that has high pH stability at both extremes and improved retention capacity compared with the conventional alkyl bonded phases, hence better resolution.  
         SUMMARY OF THE INVENTION  
         [0011]    The aforementioned needs are satisfied by several aspects of the present invention. One aspect of the present invention provides a method for separating a plurality of different chemicals in a sample comprising said different chemicals. The method comprising the steps of: preparing a stationary phase composition comprising a polymer-based chromatographic support which comprises chemically stable and porous particles having a surface, the surface of said particles having hexyl-phenyl bonded thereto; packing said polymer-based chromatographic support inside a column; injecting said sample comprising a plurality of different chemicals into a first end of the column; and applying liquid flow into said first end of said column, thereby forcing said sample through said column and out of a second end of said column, whereby said different chemicals come out of said second end of said column at different times.  
           [0012]    Another aspect of the present invention provides a method of using a composition in reversed-phase liquid chromatography as stationary phase to separate a plurality of different chemicals in a sample comprising said different chemicals, the composition comprising a polymer-based chromatographic support which comprises chemically stable and porous particles having a surface, the surface of said particles having hexyl-phenyl bonded thereto.  
           [0013]    More further aspect of the present invention provides a composition for use as a stationary phase in reversed-phase liquid chromatography, comprising a polymer-based chromatographic support which comprises chemically stable and porous particles having a surface, wherein said surface of said particles has two or more different alkyl-phenyls bonded thereto, wherein said alkyl-phenyls have from five to ten methylene groups in their alkyl groups. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0014]    [0014]FIG. 1 shows schematic reaction for synthesis of an alkyl-phenyl bonded silica gel.  
         [0015]    [0015]FIG. 2A and 2B show the effect of pH on the stability of hexyl-phenyl bonded silica gel.  
         [0016]    [0016]FIG. 3 is a plot for showing selectivities of alkyl (C5, C8, and C18) and alkyl-phenyl (C3Ph and C6Ph) bonded phase for alkylbenzenes in 80% acetonitrile.  
         [0017]    [0017]FIG. 4A and 4B show the selectivities of pentyl (C5) and hexyl-phenyl (C6Ph) bonded phases for alkylbenzenes in 70% and 80% aqueous acetonitrile respectively.  
         [0018]    [0018]FIG. 5 is a plot for comparing the selectivities of octyl (C8) and hexyl-phenyl (C6Ph) bonded phases for alkylbenzenes and polyaromatic hydrocarbons (PAH).  
         [0019]    [0019]FIG. 6 shows a result of separating antibacterial drugs hexyl-phenyl (C6Ph), propyl-phenyl (C3Ph), octyl (C8), and octadecyl (C18) bonded phases. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0020]    We analyzed the molecular and chromatographic properties of phenyl bonded phases, attached to silica gel via one to twelve methelene groups by calculating the log P values thereof. The calculation of log P value is disclosed in R. F. Rekker, “The hydrophobic fragmental constant”, Elsevier, Amsterdam, (1997) which is hereby incorporated herein by reference. The calculated log P values based on Rekker&#39;s fragmental constants for different functional groups are summarized in TABLE 1.  
         [0021]    The log P values of bonded phases indicate the retention capacity and the chemical stability. The log P values of chemically unstable phases, such as propyl-phenyl, propyl-amino, propyl-cyano, and butyl-groups are small, and these phases show lesser retention and lower stability. The larger log P values mean higher hydrophobicity, and lead to longer retention and higher stability of the bonded phase.  
         [0022]    The low retentivity and stability of the conventional phenyl phases are the result of short alkyl chains, e.g. methyl, ethyl, and propyl used in attaching the phenyl group on the surface of the silica gel. Because of this low hydrophobicity, the unique selectivity of the phenyl phases cannot be fully realized. The reason for the instability of the short chain bonded phase can be understood from the effect of alkyl chain length on hydrogen bonding capacity of alkanols. Up to four methylene units (butyl groups) the hydrogen bonding capability of hydroxy groups is affected by the alkyl chain length, but the longer alkyl chain does not further affect the hydrogen bonding capability. As such, the butyl bonded phase is not stable in high pH solutions, but the stability of pentyl bonded phases is equivalent to octyl and octadecyl bonded phases.  
         [0023]    The analysis of the properties of bonded phases of silica gels from their molecular masses and log P values, as seen in Table 1, indicate that alkyl-phenyl bonded phases having 4 to 10 methylene groups in their alkyl group should theoretically lead to better stability and selectivity than propyl-phenyl, ethyl-phenyl, or methyl-phenyl bonded phase. Preferably, alkyl-phenyl bonded phase, having five to ten methylene groups in the alkyl group have high stability and selectivity. Most preferably, hexyl-phenyl has the ideal stability and selectivity.  
         [0024]    We prepared the alkyl-phenyl bonded silica gels and tested its stability and separation power compared to the other alkyl bonded phases. According to the present invention, alkyl-phenyl bonded silica gels can be synthesized using a variety of silylating reagents. The synthesis was carried out by reacting a mono- , di- , or tri- functional alkyl-phenylsilane with the silanol groups of silica gels in a suitable media under high temperature as depicted in FIG. 1. The functional groups are good leaving group such as halogens, methoxy, ethoxy, etc, and the alkyl-phenylsilane having one to three leaving groups, hereinafter, is referred to as a “reactive alkyl-phenylsilane”.  
         [0025]    The important aspect of the present invention is that the alkyl-phenyl bonded phases of the present invention shows chemical stability over extended pH range of 1.5 to 10.00 and has unique selectivity for the separation of organic molecules compared to other alkyl bonded phases. The stability and selectivity of alkyl-phenyl bonded phase of the present invention are not limited by the use of various polymer-based chromatographic supports such as silica gel, polystyrene-divinylbenzene copolymer, polyhydroxymethacrylate, cellulose, etc. Additionally, various alkyl-phenyl bonded phases can be mixed to provide a mixed phase while maintaining their own selectivities and retaining capacities. In sum, the alkyl-phenyl bonded phases of the present invention have many advantages when applied to perform HPLC, in terms of its inertness (low silanophyllic activity), extended pH stability and unique selectivity.  
       EXAMPLE 1  
     Preparation Of Alkyl-Phenyl Silica Gels And Columns Packed Therewith  
       [0026]    In a typical experiment, twenty grams of the silica gel were washed with 0.1 N hydrochloric acid (HCl) on a filter funnel followed by distilled and dionized water till the pH of wash was neutral (nearly 7.0). The silica gel was then washed with acetone and dried in an oven at 80° C. under vacuum for 10 hours.  
         [0027]    A dry 500 mL capacity round bottom flask was set up in a fume hood, and 20 grams of hexyl-phenyl methyldichlorosilane as a silylating agent for obtaining hexyl-phenyl bonded phase and 200 mL of dry toluene were added to it. The two were mixed thoroughly and the twenty grams of dried silica gel were added to the reaction mixture while continuously stirring the mixture. The flask was then attached to a condenser with water circulating through it and heated to boiling using a heating mantle. After allowing the reaction to proceed for 18 hours, the flask was cooled and the silica gel which had hexyl-phenyl group on the surfaces was filtered. The hexyl-phenyl bonded silica gel was then washed with 100 mL dichloromethane, 100 mL methanol and 100 mL acetone and dried under vacuum at 80° C. A similar reaction and procedure were carried out to prepare propyl-phenyl bonded silica gel.  
         [0028]    After the bonding step, the unreacted silanol groups on the surface of the silica gel were blocked by an end-capping step as follows: the bonded silica gel was added to a mixture of 40 mL of trimethylchlorosilane and 200 mL of toluene in a round bottom flask and reacted for 18 hours. At the end of the reaction, the silica gel was subjected to the washing/drying procedures as described earlier in the bonding step.  
         [0029]    The end-capped hexyl-phenyl and propyl-phenyl bonded silica gels were packed into two individual 150 mm L×4.6 mm I.D. columns for the chromatographic evaluation.  
         [0030]    Similar reactions and procedures are carried out to prepare other alkyl-phenyl bonded silica gels which have 4, 5, 7, 8, 9, or 10 methylene groups in their alkyl groups, to end-cap unreacted silanols on their surfaces, and to provide columns inside of which is packed with the alkyl-phenyl bonded silica gels.  
         [0031]    All the organic solvents used in the reaction were obtained from J. T. Baker (Phillipsburg, N.J. USA). The organosilane reagents were from Silar Laboratories (Wilmington, N.C. USA). The silica gel used was obtained from Phenomenex, Inc. (Torrance, Calif. USA) and had the following specifications: 5 μm dp, with 100 Å average pore diameter and 400 m 2 /g surface area.  
       EXAMPLE 2  
     Inertness And Chemical Stability Of Alkyl-Phenyl Bonded Phases  
       [0032]    The inertness and the chemical stability of the synthesized hexyl-phenyl bonded silica gel were studied by looking at the retention factors and peak shapes of pyridine, benzoic acid, 8-hydroxyquinoline, and naphthalene disclosed in T. Hanai, Encyclopedia of Analytical Science (1995), Academic Press, London, p. 2558 - 1567 which is hereby incorporated herein by reference. The retention factor, in terms of measured parameters, is “k=(t R −t O )/t O ”: where t R  is retention time of the measured peak; and to is retention time of non-retained component.  
         [0033]    The plots 20 and 25 in FIGS. 2A and 2B show chemical stability of hexyl-phenyl bonded phase in acidic and basic solutions. The lines 21 and 26 indicate retention time (t R ) for toluene, and the lines 22 and 27 represent retention factors (k) for toluene in acidic and basic solutions respectively. The lines 23 and 28 indicate peak asymmetry for toluene, and the lines 24 and 29 for pyridine. Asymmetry is a factor describing the shapes of chromatographic peaks, and the factor is the ratio of the distance between the peak apex and the back side of the chromatographic curve and the front side of the curve at 10% peak height. As shown in FIGS. 2A and 2B, the retention time and retention factor for toluene was almost constant in continuous operation of more than 2,500 hours (more than 60,000 column volumes) both in 10 mM sodium phosphate buffer (pH 10) and 0.1% trifluoro acetic (pH 1.5) solutions. In addition, the peak shapes of toluene and pyridine were almost symmetric. The surface was also inert for trace metal sensitive test using 8-hydroxyquinoline, thereby showing a low level of metal contamination.  
       EXAMPLE 3  
     Retention Capacity Of Alkyl-Phenyl Bonded Phases  
       [0034]    The retention capacities of alkyl-phenyl bonded silica gels were compared from the retention factors for alkylbenzenes and polycyclic aromatic hydrocarbons (PAH). The log k values of benzene, 16 PAHs, and 10 alkylbenzenes for different bonded phases are listed in Table 2.  
         [0035]    The log k values of alkylbenzenes were measured by reversed-phase liquid chromatography in aqueous 80% acetonitrile at 40° C. The log k values were used for the analysis of hydrophobicity of bonded phases using van der Waals volumes of analytes as the standard. The relations between log k values and van der Waals volumes of alkylbenzenes in different bonded phases are shown in FIG. 3. The relations indicate that the retention capacities related to hydrophobicity of hexyl-phenyl and propyl-phenyl bonded phases (respectively C6Ph and C3Ph) were less than that of the pentyl bonded phase (C5). The relation between their slope and alkyl chain length was: Alkyl chain length=4.321×(slope) −22.729, r 2 =0.997 (n=3).  
         [0036]    The retention capacity of synthesized hexyl-phenyl bonded phase (C6Ph) for alkylbenzenes in 70% aqueous acetonitrile was equivalent to that of pentyl bonded phase (C5) as shown in FIG. 4A; however, that for polycyclic aromatic hydrocarbons (PAH) on C6Ph was equivalent to that of octyl bonded phase (C8) as shown in FIG. 5. As shown in FIG. 4B, pentyl bonded phase (C5) is slightly more retentivity than hexyl-phenyl bonded phased (C6Ph) in 80% aqueous acetonitrile. Advantageously, the difference in retentivity seen with different solvents can be used to effect better separation simply by changing solvents.  
         [0037]    When the retention time of alkylbenzenes was used as the standard, the ratios of the slopes for the plot of log k values and van der Waals volumes indicated the difference of retention capacity of these bonded phases (calculated from FIG. 5). The retention capacity for alkylbenzenes was 1.27 (6.52/5.117) times greater than that for PAH on C6Ph phase. The retention capacity for alkylbenzenes was 1.73 (7.840/4.531) times greater than that for PAH on C8 phase. This means that PAHs were retained 1.36 (1.73/1.27) times on C6Ph phase than on C8 phase.  
       EXAMPLE 4  
     Selectivity of Hexyl-phenyl Bonded Phase for Antibacterial Drugs  
       [0038]    The selectivities of the alkyl-phenyl and alkyl bonded phases were examined for the separation of antibacterial drugs. The mixtures of carbodox, thiamphenicol, furazolidine, oxolinic acid, sulfadimethoxine, sulfaquinoxiline, nalidixic acid and piromidic acid were chromatographed using potassiumdihydrogen phosphate and acetonitrile mixtures. The total elution time was about 20 minutes in C3Ph, C6Ph, C8, and C18 bonded phases as shown in FIG. 6. The perfect resolution between sulfadimethoxine and sulfaquinoxiline was achieved only on C6Ph phase, and the separation of these two compounds was not possible on C8 and C18 phases.  
         [0039]    In conclusion, such selective retention capability of phenyl bonded phases can expand the separation power in reversed-phase liquid chromatography. An eluent for C8-bonded phase can be easily applied to C6Ph phase for the further separation. Such two-dimensional chromatograms can speed up the analysis of complex mixtures. Above results also indicate that hexadecyl-phenyl bonded phase will be a compatible phase to C12 bonded phase for improving separation power in reversed-phase liquid chromatography.  
         [0040]    The computer used for the molecular calculations were Macintosh 8100/100 running the CAChe™ program including ProjectLeader™ from Sony-Tektronix (Tokyo). Rlog P values were calculated by a method based on that proposed by Rekker. The van der Waals volumes were calculated by MOPAC-Blog P program provided by Sony-Tektronix. The molecules were first optimized by molecular mechanics calculation, and optimized again by MOPAC, then their van der Waals volumes and log P values were obtained with the Blog P program disclosed in CAChe Manual, Sony-Tektronix, (1995) which is hereby incorporated herein by reference. Properties for the calculation were selected according to the manual from CAChe Scientific.  
         [0041]    The liquid chromatograph was a model HP1090 from Hewlett-Packard (Palo Alto, Calif. USA), and the chemically-bonded silica gel columns, —C18, —C8 and —C5, —C6Ph, —C3Ph, 15 cm×4.6 mm I.D., were from Phenomenex Inc. (Torrance, Calif. USA). Chemicals used were from Aldrich and ChemService. HPLC grade acetonitrile and water were from Fisher Scientific (Tustin, Calif. USA).  
                                                   TABLE 1                           Log P Values of Bonded Groups                CHEMICAL NAME   STRUCTURE   LOG P                            Phenyl   (—Ph)   1.89           Methyl-phenyl   —CH 2 Ph   2.42           Ethyl-phenyl   —(CH 2 ) 2 Ph   2.95           Propyl-phenyl   —(CH 2 ) 3 Ph   3.46           Butyl-phenyl   —(CH 2 ) 4 Ph   3.99           Hexyl-phenyl   —(CH 2 ) 6 Ph   5.05           Octyl-phenyl   —(CH 2 ) 8 Ph   6.09           Decyl-phenyl   —(CH 2 ) 10 Ph   7.15           Dodecyl-phenyl   —(CH 2 ) 12 Ph   8.21           Methyl or C1   —CH 3     0.70           Ethyl or C2   —CH 2 CH 3     1.40           Propyl or C3   —CH 2 CH 2 CH 3     2.10           Butyl or C4   —CH 2 CH 2 —CH 2 CH 3     2.93           Octyl or C8   —(CH 2 ) 7 CH 3     4.41           Octadecyl or C18   —(CH 2 ) 17 CH 3     9.01           Cyano   —(CH 2 ) 3 CN   0.52           N-Methyl-N-cyanoethyl   —N(CH 3 )(CH 2 ) 2 CN   −1.46           Propyl amino   —(CH 2 ) 3 NH 2     0.16           Butyl amino   —(CH 2 ) 4 NH 2     0.69                      
 
         [0042]    [0042]                                                                                                                                                                                                                   TABLE 2                           van der Waal&#39;s Volumes and Log K Values       of PAH and Alkylbenzenes                log kin 70% CH3CN                    VWV                               PAH   A3/mole   C5   C8   C18   C3Ph   C6Ph                    Benzene   83.789   0.209   0.281   0.340   0.167   0.230           Naphthalene   127.598   0.410   0.486   0.582   0.305   0.470       Acenaphthylene   144.969   0.450   0.540   0.663   0.364   0.521       Acenaphthene   150.675   —   0.629   0.814   0.447   0.616       Fluorene   161.639   0.527   0.629   0.794   0.436   0.602       Phenanthrene   171.286   0.538   0.660   0.854   0.468   0.655       Anthracene   171.486   0.562   0.688   0.894   0.490   0.679       Pyrene   187.603   0.614   0.777   1.038   0.553   0.778       Fluoranthene   188.723   0.599   0.747   0.989   0.535   0.746       Chrysene   214.558   0.673   0.842   1.160   0.635   0.869       Benzo(a)anthracene   215.641   0.692   0.860   1.177   0.647   0.878       Benzo(a)pyrene   231.048   0.755   0.071   1.375   0.733   1.009       Benzo(k)fluoranthene   232.418   0.741   0.959   1.347   0.726   0.982       Benzo(b)fluoranthene   232.462   0.741   0.943   1.325   0.714   0.970       Benzo(g,h,i)perylene   247.198   0.822   1.067   —   0.799   1.118       Dibenzo(a,h)anthracene   259.435   0.803   1.032   1.479   0.806   1.071       Indeno(1,2,3 cd)pyrene   248.683   0.822   1.067   —   0.803   1.102                        log k in 70% CH3CN   log k in 80% CH3CN                VWV                                               Alkylbenzene   A3/mole   C5   C8   C18   C3Ph   C6Ph   C5   C8   C18   C3Ph   C6Ph                    Toluene   100.456   0.312   0.396   0.489   −0.253   0.326   0.170   0.225   0.267   0.049   0.137       Ethylbenzene   117.067   0.417   0.513   0.628   0.348   0.428   0.258   0.324   0.394   0.121   0.215       Propylbenzene   133.897   0.537   0.644   0.800   0.449   0.538   0.362   0.442   0.548   0.196   0.303       Butylbenzene   150.713   0.653   0.776   0.963   0.548   0.648   0.462   0.561   0.702   0.274   0.391       Pentylbenzene   167.564   0.772   0.908   1.134   0.646   0.758   0.567   0.680   0.858   0.350   0.480       Hexylbenzene   184.315   0.894   1.043   1.310   0.747   0.871   0.679   0.806   1.021   0.430   0.572       Heptylbenzene   201.171   1.019   1.181   1.491   0.851   0.985   0.794   0.937   1.188   0.512   0.667       Octylbenzene   217.884   1.144   1.318   1.673   0.947   1.095   0.906   1.063   1.356   0.589   0.757       Nonylbenzene   234.706   1.271   1.457   —   1.046   1.204   1.022   1.193   1.528   0.668   0.849       Decylbenzene   251.540   1.400   —   —   1.146   1.313   1.139   1.324   1.697   0.748   0.941