【连载】填料粒径及其分布对色谱性能的影响

企业   2024-09-25 17:20   上海  

目前,以高效液相色谱(HPLC)和超高效液相色谱(UHPLC)为代表的液相色谱已成为制药[1,2,3]、食品[4]、环境[5]和法医学[6,7,8,9,10]等行业实现快速分离的主要分析手段。早在1903年Tswett博士就在华沙的一次当地会议上展示了他的色谱分离结果[11],但是,液相色谱花费了六十多年的时间、直到20世纪70年代,才以高效液相色谱(HPLC)技术走出了研究实验室的高墙[12]

在此期间,有两个因素严重阻碍了液相色谱的发展步伐:填料颗粒和仪器。即便诺贝尔奖得主Martin和Synge[13]在1941年发表的分配色谱法文章中强调了这两个因素 ,但情况仍没有太大改观。纵观色谱技术的发展史,与液相色谱密切相关的两类专门仪器,自动化氨基酸分析仪和凝胶过滤色谱仪,早在1958年[14,15]和1963年初[16]就已经分别上市了,因此可以说,当时的大粒径 (>100μm)、不规则形状、全多孔的填料是阻碍液相色谱发展的主要原因。

为了克服上述填料的缺点,液相色谱的先驱们创造了由实心核、聚合物包覆层[17,18]或纳米级球形硅胶颗粒多孔层[19,20,21]的填料,这类填料曾被命名为多层孔微球、表面孔隙可控颗粒、核壳型颗粒(SPP)及薄层多孔颗粒。与当时通用的不规则全多孔硅胶、氧化铝颗粒以及离子交换树脂相比,其理论塔板数有了显著提高[22], 由此类微球衍生出来的商业化的液相色谱产品超过了20多个[23]

01

填料颗粒的表征

经过长期的研究,色谱填料颗粒的物理性质和色谱性能之间的相关性已经得到很好地证明[24,8,10],这些参数可以总结如下:

粒径(dp)及其分布(σp)

1941年,Martin和Synge就已经指出了粒径对色谱分离的影响,粒径愈小,柱效愈高,较小粒径的填料成为快速高效分析的必然选择。然而,粒径的减小对色谱柱所引起的压力,即柱压(ΔP),有着重大影响,其关系可用下面的公式来表示 [25]

Ø:柱流阻力  L:柱长(cm)  η:流动相粘度(cP)

由公式1可见,填料粒径减小一半,色谱柱的柱压将升高4倍,因此在2004年前,在传统HPLC仪器上应用粒径小于2μm的填料几乎是不可能的。

公式1不仅可以用来预测色谱柱的柱压,也是材料科学家们用来设计色谱材料的一个非常有力的工具。

例如,在核壳型填料问世之前,Kirkland先生曾经使用该公式,来确定能够用于普通HPLC仪器上的核壳型硅胶的最小粒径。具体是:在HPLC上运行50×4.6mm色谱柱、压力极限为400bar、粘度为1.6cP的典型流动相、流速在1.0mL/min[26]的方案下,利用公式1可以推算出核壳型硅胶填料颗粒的最小粒径为2.7μm。

粒径对色谱分析的第二个影响是渗透性(Kpm),它对色谱峰展宽有着很大的影响。正如公式2[24]所示,随着粒径变小,Kpm迅速降低。

εi:间隙孔隙度

球形颗粒的填充床比不规则颗粒[27]的填充床具有更好的渗透性。对于传统的全孔型硅胶(TPP)色谱柱,柱床的间隙孔隙率( ε)为0.4 [24] 。然而,与之不同的是,核壳型的多孔壳层是被一层层堆积出来的,而且不同方法之间的形貌可能有很大的差异。

由于这一特性,再加上不同的测量方法,有关间隙孔隙率的报道大相径庭,从 0.191(0.05-μm外壳厚度)[28]到0.405[29],0.432[30,31]和0.583- 0.596[32]。核壳型填料渗透性的修正公式Kpm可表达为如下公式[33]

由公式3可知,当两个色谱柱具有相同的间隙孔隙度εi时,具有较低的总孔隙度εT的色谱柱的渗透率更大。因此,当将核壳型色谱柱与粒径相近的全孔型色谱柱相比较时,较低的εT(15-25%)具有较高的Kpm,SPP(20-30%)。

而由于Kpm,SPP的增加,分析物在核壳型色谱柱中的滞留时间就更短,因而发生纵向扩散的时间比全孔型色谱柱要短,从而导致van Deemter或Knox公式[34]中的B项明显减小,结果是柱效增加。

van Deemter 公式

Knox 公式


与粒径相关的另一个参数是粒度分布(σp), 它对传质效率和van Deemter公式A项[35],以及Knox公式的A项[36]有很大影响。图1给出了:

(a) hmin随σp的变化曲线

(b) A项随σp的变化曲线

(c) Ei,min (最小分离阻抗)随σp的变化曲线

Ei定义如下:

H:踏板高度  εi:间隙度  εT:总孔隙率

Kpm定义如下:

u0:流动相线速度  ΔPsys:系统压降

在这项研究中,作者引用了来自其他文献的数据[37]。由图1可见,hmin (A,或Ei, min)与σp呈线性关系,这清晰地说明了粒径分布对柱床的影响。与全孔型硅胶(TPP)相比,所有SPP柱具有较小的σp、hmin、A和Ei,min的,显示出更高的分离效率。与σp最大的TPP相比,SPP的hmin和A降低了约40%,从而保证了SPP具有更高的柱效。

(a) hmin与sigma (粒径分布宽度(PSD))关系图

(b) 报告中研究的颗粒(完整符号)和Bille

等人[37]研究的颗粒(开放符号)的A与sigma关系图

(c) 报告中的Ei,min与sigma关系图

图1

实线表示为完整符号获得的趋势线,而虚线是相同的外推趋势线。A值的置信区间也显示在图b部分。经Cabooter[35]等人许可转载。

下期预告

孔径、孔体积、比表面积与色谱性能有什么相关性?


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作者:朱晗斐、刘德云、肖留榜、骆初平

排版:赵林

审核:骆初平

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