最近のLC／MS(液体クロマトグラフィ／質量分析装置)に関する興味は、より小さい微粒子径の液体クロマトグラフィ樹脂へ移行することによって得 られる、理論段数の向上及びMSの入り口としての優位性などに集まってきています。以前の研究では微粒子径UHPLC樹脂(<3µm) を充填したカラムと、3µmできっちり充填したカラムを、短時間で急勾配なグラジエント分析条件下において比較しますと、微粒子径UHPLC 樹脂充填カラムのピーク容量は、ほとんど改善されていませんでした。しかしカラムの感度に関しましては、同じ分析条件下においてこの二つの樹脂を比較しま すと、微粒子径UHPLC樹脂充填カラムを使用したLCの感度が非常に上がりますので、MSでの感度も大きくなります。このことは微粒子径UHPLC樹脂 充填カラムが、従来の長いカラムでより勾配をねかせたグラジエントを採用して分析するよりも、格段に高い理論段数が得られた結果から、従来のカラムに関す る有用性の論争が終わってしまいました。
MSも分離能を提供しますが、重要なアプリケーション及び非常に複雑な混合サンプルの分析の場合、マトリックスの影響を小さくし、ターゲットの同定 率を高め、感度を維持するためのLC側の分離能が必要です。今回はpH1〜12の広範囲において使用できる、高純度シリカ100%を基材としたカラムで、 MSに入る前の分離能を最大限に高め、pHと分析対象物のイオン化効率の関係においてMSでの検出感度を高めること、そして同様により小さい微粒子径の液 体クロマトグラフィ樹脂へ移行した際の可能性について述べました。
Ken Butchart and Mark Woodruff, Fortis Technologies ltd., 45 Coalbrookdale Road, Cheshire, CH64 3UG, UK;
ASMS (American Society for Mass Spectrometry) 2009, Poster
Recent interest in LC-MS has revolved around the possibility of
moving to smaller LC particles; the goal being increased efficiency and
the advantages that this can provide as the inlet to MS. Although
previous work(1) has shown that for short fast gradients small UHPLC
particles offer little or no improvement in peak capacity when compared
with well-packed 3um particle columns, they do offer improved
sensitivity under these same conditions and greater sensitivity in LC
translates to greater sensitivity in the MS.
Where small UHPLC particles do show a significant efficiency increase over tradition columns is in longer columns and shallower gradients, so much so that the debate as to their usefulness has ceased.
Whilst MS can itself provide resolution, for critical applications and highly complex mixtures LC resolution is still necessary in order to reduce matrix effects, increase target identification and maintain sensitivity.
In this poster we discuss the use of LC across the pH range, how to maximise resolution prior to MS detection and the ability to move to smaller particles as well as the implications that this has in analyte response and sensitivity in the MS detector.
By looking at the Carr equation(figure 1) we can see that the three
factors contributing to resolution are efficiency, retention and
selectivity. The variable to be utilised in UHPLC is efficiency (N) but
even this is a reasonably shallow slope in relation to what can be
achieved by the selectivity (α) term.
How can we achieve this selectivity and still maintain compatibility with MS, how does the introduction of UHPLC affect our method development and use of LC-MS?
One major contributor to selectivity is the use of pH, if we
consider the separation of Lidocaine (Figure 2), a basic molecule. At
low pH there is no retention due to its ionised form being polar, from
a MS point of view this lack of retention will lead to problems from
the matrix that the sample is in. If however we can gain sufficient LC
retention then we are able to suppress any problem arising from matrix
The use of a much higher pH leads to the basic analyte existing in its neutral state and therefore retaining by hydrophobicity (Figure 3). The MS compromise here is that the source (ESI or APCI) will have to reform an ion in order to detect with high sensitivity. Another advantage of using high pH in this instance is that the molecule can now be eluted with higher organic solvent contribution which aids the mobile phase vaporisation and leads to more sensitivity.
In Figure 4 we see the advantage of gaining good selectivity, we can move to a smaller 2.1um particle from a standard C18 3um column and gain efficiency, but in the case of the two positional isomers we have still not achieved baseline separation. Since the m/z is the same for these analytes then qualification and more importantly quantitation is made difficult with MS. Changing the selectivity with the use of a diphenyl stationary phase gives us sufficient separation that we can now afford to reduce the column length and gain more speed
If we look at a highly complex environmental sample, 135 transitions, analysed on two C18 columns we can see some more parameters for good separation and sensitivity, stronger retention on one column leading to better resolution. The other variable affecting peak height here is peak width, even on the 3um columns used here the sensitivity of sample is quite different
Are we more productive with UHPLC attached to MS and relying upon efficiency alone? We set out to ask this question: undoubtedly we can do method development in a shorter time if we can speed up our analysis. However in terms of throughput of samples serious consideration has to be given to the daily logistics of UHPLC use:
UHPLC is definitely the next evolution in chromatography, the use of
small particles offers us more efficiency which can be used to increase
speed, resolution and sensitivity. However over reliance upon the term
"efficiency" must be avoided, efficiency alone will not be sufficient
for good chromatography - selectivity plays a vital role here.
We have shown that through the correct selection of particle size, pH and phase chemistry we are able to reduce analysis time and improve our MS sensitivity and target identification. Nothing should detract from good chromatography, no matter how good the MS. If LC is not optimum then the MS will be compromised as we have demonstrated.
1. K.Butchart, et al. Int. Labmate(2007) Vol. XXXII Issue V
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