摘要

液相色谱 (LC) 微型化通常是为了提高灵

敏度，这是蛋白质组学检测低丰度肽和蛋

白质的必然要求。微型化的其他优点还包

括提高与质谱连接的效率并降低溶剂的消

耗。如今，微型化液相色谱更容易使用，

它的仪器操作和连接方式均与常规液相色

谱系统一样。这为微型化液相色谱特别是

毛细管和微流液相色谱应用于新的工作流

程和应用领域提供了更多机会。

关键词

RSLCnano、纳升液相色谱、毛细管液相

色谱、微流液相色谱、质谱、nanoViper

液相色谱微型化：我们为什么这样做？

白皮书

70814

Evert-Jan Sneekes, Laurent Rieux 和 Remco Swart

赛默飞世尔科技，荷兰阿姆斯特丹

图 1. 相同进样量在液相色谱系统中的浓度不同

Miniaturization of Liquid Chromatography: Why Do We Do It? Evert-Jan Sneekes, Laurent Rieux, and Remco SwartThermo Fisher Scientific, Amsterdam, The Netherlands

Wh

ite Pap

er 708

14

Executive Summary Miniaturization of liquid chromatography (LC) is generally done to increase sensitivity, a necessity in proteomics to detect low abundant peptides and proteins. Other benefits of miniaturization include improving the efficiency of interfacing with mass spectrometry and reduction of solvent consumption. Nowadays, miniaturized LC is much easier to use and both the instrumentation and the connections are operated in the same way as conventional-scale LC systems. This opens up opportunities to apply miniaturized LC to novel workflows and applications, especially for capillary and micro LC.

IntroductionThe development of miniaturized LC started approximately three decades ago, with the first commercial products appearing in 1987. Over time nano LC has established itself as an indispensable tool for bioanalytical research, especially in the field of proteomics, which requires the highest levels of sensitivity. However with the routine use of nano LC in proteomics laboratories, the principles behind the increase in sensitivity have sometimes been forgotten. Understanding these principles and their practical implications does allow foreseeing how miniaturized LC can benefit proteomics, bioanalysis, and beyond.

Sensitivity – Size MattersIn order to understand the increase in sensitivity from smaller inner diameter (i.d.) columns, the difference between sample concentration and sample amount must first be clear. It can be summarized as follows:

• Sample injection in LC means a volume of a certain concentration is introduced – This is a fixed amount: e.g. 1 µL of 1 pmol/µL = 1 pmol

• Upon injection, this amount is diluted in the LC volume – This creates a new concentration

• A low volume LC will generate a higher concentration for the same injected amount – Smaller i.d. columns provide more sensitivity than large i.d. columns

Figure 1 shows the same injected amount (four “peptides”) on a 4.6 mm and 75 µm i.d. column. The higher concentration in the latter is evident. The relative increase in sensitivity can be calculated using Equation 1.

KeywordsRSLCnano, Nano LC, Capillary LC, Micro LC, Mass spectrometry, nanoViper

Figure 1. Different concentration in LC system for the same injected amounts.

4.6 mm0.075 mm

2 ( ) ≈ 3800

Diameter standard columnDiameter narrow column( )

2

Equation 1. Concentration Factor

引言

微型化液相色谱技术的发展始于约三十年前，1987 年出现了首批商品化产品。随着时

间的推移，纳升液相色谱成为生物分析研究不可或缺的工具，尤其是在要求最高级别

灵敏度的蛋白质组学领域。然而，随着纳升液相色谱在蛋白质组学实验室中的日常应

用，人们有时会遗忘灵敏度提高的原理。了解这些原理及其实际影响有助于帮助人们

看到微型化液相色谱将如何为蛋白质组学、生物分析等带来益处。

灵敏度 - 尺寸因素

为了解内径较小的色谱柱会使灵敏度增加这一点，首先必须明确样品浓度和样品量之

间的差异。这可以归纳如下：

• 液相色谱的样品进样是指一定体积的某浓度样品的注入

- 这是一个固定量：例如 1 μL 1 pmol/μL = 1 pmol

• 进样时，这个样品固定量被液相色谱系统稀释

- 这将得到一个新的浓度

• 在相同进样量情况下，较小体积的液相色谱将产生较高浓度

- 与大内径色谱柱相比，内径较小的色谱柱灵敏度更高

图 1 显示了在内径分别为 4.6 mm 和 75 μm 的色谱柱上注入相同进样量（四条“肽”），

显然后者的浓度更高些。相对增加的灵敏度可以采用公式 1 计算。

公式 1. 浓度因子

Miniaturization of Liquid Chromatography: Why Do We Do It? Evert-Jan Sneekes, Laurent Rieux, and Remco SwartThermo Fisher Scientific, Amsterdam, The Netherlands

Wh

ite Pap

er 708

14

Executive Summary Miniaturization of liquid chromatography (LC) is generally done to increase sensitivity, a necessity in proteomics to detect low abundant peptides and proteins. Other benefits of miniaturization include improving the efficiency of interfacing with mass spectrometry and reduction of solvent consumption. Nowadays, miniaturized LC is much easier to use and both the instrumentation and the connections are operated in the same way as conventional-scale LC systems. This opens up opportunities to apply miniaturized LC to novel workflows and applications, especially for capillary and micro LC.

IntroductionThe development of miniaturized LC started approximately three decades ago, with the first commercial products appearing in 1987. Over time nano LC has established itself as an indispensable tool for bioanalytical research, especially in the field of proteomics, which requires the highest levels of sensitivity. However with the routine use of nano LC in proteomics laboratories, the principles behind the increase in sensitivity have sometimes been forgotten. Understanding these principles and their practical implications does allow foreseeing how miniaturized LC can benefit proteomics, bioanalysis, and beyond.

Sensitivity – Size MattersIn order to understand the increase in sensitivity from smaller inner diameter (i.d.) columns, the difference between sample concentration and sample amount must first be clear. It can be summarized as follows:

• Sample injection in LC means a volume of a certain concentration is introduced – This is a fixed amount: e.g. 1 µL of 1 pmol/µL = 1 pmol

• Upon injection, this amount is diluted in the LC volume – This creates a new concentration

• A low volume LC will generate a higher concentration for the same injected amount – Smaller i.d. columns provide more sensitivity than large i.d. columns

Figure 1 shows the same injected amount (four “peptides”) on a 4.6 mm and 75 µm i.d. column. The higher concentration in the latter is evident. The relative increase in sensitivity can be calculated using Equation 1.

KeywordsRSLCnano, Nano LC, Capillary LC, Micro LC, Mass spectrometry, nanoViper

Figure 1. Different concentration in LC system for the same injected amounts.

4.6 mm0.075 mm

2 ( ) ≈ 3800

Diameter standard columnDiameter narrow column( )

2

Equation 1. Concentration Factor

2

光学检测器（如紫外检测器）和电喷雾质谱仪属于浓度敏感型仪器。它们的信号与从色谱柱洗

脱峰中样品的浓度成正比。

图 2 绘制了不同直径的色谱柱较直径为 4.6 毫米的色谱柱的灵敏度相对增加（按公式 1 计算）。

从图中可看出，内径最小（50 μm 和 75 μm）的色谱柱，其灵敏度在理论上可提高几千倍。

然而，插图显示色谱柱内径从 4.6 mm 变为 1 mm 时，灵敏度已能得到显著提高（约 20 倍）。在

一些不要求绝对灵敏度，但微型化可以提供其它益处，例如与质谱轻松连接，减少溶剂用量（见

计算示例）和废液处置量并降低成本的情况下，这些色谱柱内径范围完全适合实际应用。

减少溶剂用量：

设想以如下参数在内径为 4.6mm 的色谱柱上进行分析：

- 流速为 1.0 mL / min

- 70％ 流动相 A

- 2 公升溶剂瓶

所有流动相 A 将在两天内耗尽。

采用微流速液相色谱法以 50 μL/min 的流速运行本应用，500 mL 的瓶子可维持一周。

提高灵敏度

提高灵敏度

色谱柱内径 (mm)

色谱柱内径 (mm)

图 2. 一系列内径的色谱柱相较于内径为 4.6 mm 的色谱柱灵敏度的相对提高倍数

3

3UHPLC Instrumentation for Nano, Capillary and Micro LC This calculated sensitivity increase is actually a scale factor between the conventional and miniaturized LC application, with flow rate as the best example. Typically, the flow rate in conventional LC (4.6 mm i.d.) is 1000–1200 µL/min and in nano LC (75 µm i.d.) 0.250–0.300 µL/min. The ratio of 4000 reflects the scale factor between a 4.6 mm i.d. and a nano LC column and will result in (approximately) the same linear velocity through both columns.

The Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLCnano system (Figure 3) has been designed to deliver the very low flow rates and very high pressures required for modern nano LC. The UltiMate 3000 RSLCnano system can deliver flow rates as low as 20 nL/min* and up to 50 µL/min at UHPLC pressures, making it the perfect instrument for nano, capillary, as well as micro LC.1 The module has a built-in column compartment to support a range of applications, while minimizing the physical distance between the various LC elements. This allows connection tubing to be as short as possible.

Another important aspect to connection tubing is the inner diameter. The connection tubing in nano LC (20 µm) is roughly 10 times smaller in inner diameters. Usually in capillary and micro LC, 50 and 75 µm i.d. tubing is used, respectively.

The best results are obtained if the correct tubing is also properly connected. Thermo Scientific™ Dionex™ nanoViper™ fittings provide a factory-made, fingertight UHPLC connection, which is virtually dead-volume free. nanoViper fittings come integrated on the connection tubing and columns and allow for tool-free assembly, without the risks of introducing dead volume or breaking capillaries upon connection.

*Lowest flow rates are only available upon request

Figure 3. UltiMate 3000 RSLCnano system, interior view, and nanoViper fitting.

纳升级、毛细管级和微流液相色谱的超高效液相色谱仪 (UHPLC)

计算得出的灵敏度增量实际上是常规和微型化液相色谱应用之间的比例因子，而流速是最好的例子。通常情

况下，常规液相色谱中的流速（内径为 4.6 mm）为 1000-1200μL/min，纳升级液相色谱（内径为 75μm）为 0.250-

0.300μL/min。4000 的比率反映内径为 4.6 mm 的色谱柱与纳升级液相色谱柱之间的比例因子，并将导致两个色

谱柱的线性速度（大致）相同。

Thermo ScientificTM DionexTM UltiMateTM 3000 RSLCnano 系统（图 3）旨在为现代纳升液相色谱提供所需的非常低的流

速和非常高的压力。UltiMate 3000 RSLCnano 系统在 UHPLC 压力条件下，提供的流速可低至 20 nL/min*、高至 50

μL/min，使其成为可实现纳升级、毛细管级和微流速液相色谱分析的完美仪器。1 模块具有内置柱温箱，可支

持一系列应用，同时可最大限度地缩短各种元件之间的物理距离。这可使连接管尽可能地缩短。

连接管的另一个重要方面是管路内径。纳升级液相色谱 (20 μm) 的连接管内径大约比常规管路内径小 10 倍。

通常，毛细管级和微流速液相色谱分别采用内径为 50μm 和 75 μm 的连接管。

如果正确连接合适的管路，就会得到最好的结果。Thermo ScientificTM DionexTM nanoViperTM 接头系统可提供厂制、

手紧 UHPLC 接头，零死体积。nanoViper 接头和连接管及色谱柱集于一体，无需工具即可组装，且没有引入死

体积或连接时毛细管断裂的风险。

* 要求时才会提供最低流速

图 3. UltiMate 3000 RSLCnano 系统、内视图和 nanoViper 接头

44 Miniaturized LC – Fields of Application

Miniaturized LC is often divided in three categories or flow rate ranges: nano, capillary, and micro LC. Nano LC refers to applications with flow rates below 1000 nL/min, capillary LC encompasses applications in the low µL/min flow rate range (1–10 µL/min), while micro LC covers the use of flow rates above 10 µL/min.

Examples of typical applications are shown next for each flow range. The first example and most common nano LC application is in proteomics discovery workflows. Here, sample limitation is the major reason for nano LC. In the second example, capillary LC is applied to the targeted quantification of peptides of a complex matrix. Sample availability might be higher, but throughput is essential. Capillary LC offers the best of both worlds. For the third example, micro LC is applied to increase throughput in the analysis of a monoclonal antibody (MAb) digest. Here sample availability is not the limiting factor, but the added sensitivity with robustness is of value. Lastly, an example outside of bioanalysis is provided.

In proteomics, researchers face a double challenge. The proteins of interest are present at low concentration and sample volumes are small, resulting in extremely low amounts. Additionally, samples are extremely complex. These factors led to the development of nano LC systems and columns with very high separation power enabling unique applications as shown in Figure 4. Köcher et. al. have performed a similar experiment, where they on average identified 2516 proteins in the tryptic HeLa digest using an 8 h gradient on a 50 cm column.2

Figure 4. High resolution separation of E.coli digest on a 75 µm i.d. × 50 cm nano LC column with a 10 hour gradient.

Rel

ativ

e A

bund

ance

微型化液相色谱 - 应用领域

微型化液相色谱通常可分为三类或三种流速范围：纳升级、毛细管级和微流速液相色谱。纳升级

液相色谱是指流速低于1000 nL/min的应用，毛细管级液相色谱包含低流速范围 (1–10 μL/min) 的应用，

而微流速液相色谱涵盖流速超过 10 μL/min 的应用。

以下是各流速范围的典型应用示例。第一个也是最常见的示例，纳升级液相色谱应用于蛋白质组

学发现工作流程中。在这里，样品量的限制是使用纳升级液相色谱的主要原因。在第二个例子

中，毛细管级液相色谱被应用于复杂基质中目标肽的定量分析。样品可能较易得到，但通量也很

关键，毛细管级液相色谱可做到二者兼顾。对于第三个示例，微流速液相色谱被用来提高单克隆

抗体 (MAb) 消化物分析中的通量。在这里，样品量不再是限制因素，但增加的灵敏度和系统耐用

性很有价值。最后，给出一个非生物分析的例子。

在蛋白质组学研究中，研究人员面临着双重挑战。待研究的蛋白质浓度低且样品体积小，导致

含量极低。此外，样品非常复杂。这些因素促使开发出纳升级液相色谱系统和具有非常高分离

能力的色谱柱，确保特殊应用，如图 4 所示。科赫尔 (Köcher) 等人进行了类似的实验，他们通过

在 50 cm 的色谱柱上进行 8 小时梯度洗脱，从而在胰蛋白酶消化的 HeLa 细胞中平均鉴别出 2516

个蛋白质。2

相对丰度

时间（分钟）

图 4. 在 75 μm（内径）×50 cm 的纳升级液相色谱柱上经 10 小时梯度洗脱后

高分辨率分离的大肠杆菌消化物

5

5The next example is the capillary LC application in a targeted quantitative workflow. In this case, capillary LC was chosen to increase throughput, robustness, and ease-of-use, while retaining great sensitivity. A yeast digest spiked with isotopically-labeled peptides was separated on a 300 µm i.d. column. The sample was separated in 30 minutes using a 4 µL/min flow rate and coupled to a Thermo Scientific™ TSQ Vantage™ triple quadrupole mass spectrometer using the standard HESI-II interface. Detection was performed in SRM mode down to 10 amol (Figure 5). More details can be found in Thermo Scientific application note 583.3

10Time (min)

Rel

ativ

e A

bund

nace

100

0

RT: 15.18AA: 2266011

NL: 2.87E5

NL: 2.75E4

NL: 3.79E3

NL: 1.06E3

NL: 7.37E2

NL: 7.72E1Yeast matrixblank

0.01 fmol

0.1 fmol

1 fmol

10 fmol

100 fmol

11 15 17 1816141312 2019

RT: 15.15AA: 8768

RT: 15.16AA: 223785

RT: 15.14AA: 27169

RT: 15.18AA: 5565

100

0100

0100

0100

0100

0

Figure 5. Extracted chromatograms of the peptide SAAGAFGPELSR spiked into 500 ng of yeast-digest matrix at different concentration levels.

The following micro flow example is of particular relevance for high efficiency and high throughput peptide mapping applications in the biopharmaceutical industry. In this specific case, a MAb digest was first separated by a generic 30 minute gradient at 6 µL/min. In two consecutive steps the flow rate was raised to 15 µL/min, while the gradient was shortened simultaneously. Figure 6 shows how the analysis time could be reduced by a factor of 3 without compromising the data quality. Raising the flow in a similar fashion on a standard-bore LC column would probably complicate the interfacing with the mass spectrometer and require eluent replacement twice a day.

Figure 6. Speeding up the analysis of a Lys C MAb digest on a 300 µm i.d. × 15 cm Thermo Scientific™ Acclaim™ PepMap™ RSLC column.

下一个例子是毛细管级液相色谱在目标定量工作流程中的应用。在本例中，选择毛细管级液相色

谱来增加通量、提高耐用性和易用性，同时保持很高的灵敏度。在内径为 300 μm 的色谱柱上分离

出添加同位素标记肽的酵母消化物。样品在 30 分钟内以流速 4μL/min 被分离，并连接到采用标准

HESI-II 接口的 Thermo ScientificTM TSQ VantageTM 三重四极杆质谱仪，在 SRM 模式下进行检测，检测限

达到 10 amol（图 5）。更多详情可参阅 Thermo Scientific 的应用报告 583。3

图 5. 按不同浓度向 500 ng 酵母消化物基质中加入肽 SAAGAFGPELSR 的提取色谱图

5The next example is the capillary LC application in a targeted quantitative workflow. In this case, capillary LC was chosen to increase throughput, robustness, and ease-of-use, while retaining great sensitivity. A yeast digest spiked with isotopically-labeled peptides was separated on a 300 µm i.d. column. The sample was separated in 30 minutes using a 4 µL/min flow rate and coupled to a Thermo Scientific™ TSQ Vantage™ triple quadrupole mass spectrometer using the standard HESI-II interface. Detection was performed in SRM mode down to 10 amol (Figure 5). More details can be found in Thermo Scientific application note 583.3

10Time (min)

Rel

ativ

e A

bund

nace

100

0

RT: 15.18AA: 2266011

NL: 2.87E5

NL: 2.75E4

NL: 3.79E3

NL: 1.06E3

NL: 7.37E2

NL: 7.72E1Yeast matrixblank

0.01 fmol

0.1 fmol

1 fmol

10 fmol

100 fmol

11 15 17 1816141312 2019

RT: 15.15AA: 8768

RT: 15.16AA: 223785

RT: 15.14AA: 27169

RT: 15.18AA: 5565

100

0100

0100

0100

0100

0

Figure 5. Extracted chromatograms of the peptide SAAGAFGPELSR spiked into 500 ng of yeast-digest matrix at different concentration levels.

The following micro flow example is of particular relevance for high efficiency and high throughput peptide mapping applications in the biopharmaceutical industry. In this specific case, a MAb digest was first separated by a generic 30 minute gradient at 6 µL/min. In two consecutive steps the flow rate was raised to 15 µL/min, while the gradient was shortened simultaneously. Figure 6 shows how the analysis time could be reduced by a factor of 3 without compromising the data quality. Raising the flow in a similar fashion on a standard-bore LC column would probably complicate the interfacing with the mass spectrometer and require eluent replacement twice a day.

Figure 6. Speeding up the analysis of a Lys C MAb digest on a 300 µm i.d. × 15 cm Thermo Scientific™ Acclaim™ PepMap™ RSLC column.

相对丰度

时间（分钟）

以下微流速示例对生物制药行业中高效率和高通量肽谱图匹配应用特别适用。在这个特定例子中，

首先以 6 μL/min 流速通过 30 分钟梯度洗脱分离单克隆抗体消化物。在两个连续步骤中，流速升高

至 15μL/min，同时梯度缩短。图 6 显示了在不降低数据质量的情况下分析时间如何缩短到 1/3。以

类似的方式提高标准粒径填料液相色谱柱上的流速，可能会使色谱系统与质谱仪的连接变得复杂，

并需要每日更换两次流动相。

图 6. 在 300 μm（内径）×15 cm 的 Thermo ScientificTM AcclaimTM PepMapTM RSLC 分析柱上

加速分析单克隆抗体 Lys C 消化物

免费服务热线： 800 810 5118 400 650 5118 ( 支持手机用户 )

赛默飞世尔科技（中国）有限公司

白皮书

70814

WP70817_E 09/13S

最后，Xiang He 等人展示了微流速液相色谱在法医毒理学等领域取得的可喜成果。4 建立了简便、

高灵敏的方法，可量化分析生物样品中的大麻素，满足法医毒理学的需要。这种方法在设计和循

环时间上与典型的标准粒径填料应用类似，但灵敏度更高，可以分析进一步稀释的样品。法医毒

理学者可将这种多用途的工作流程应用于其他常见的非法 / 滥用药物定量分析。试剂的消耗量也

会显著降低 90％以上，并降低质谱仪的维护频率。

结论

液相色谱仪的微型化通常仅被认为与蛋白质组学研究所要求的可提供更高灵敏度的纳升级液相色

谱相关。但是，毛细管级和微流速液相色谱已大幅度提高了灵敏度。此外，在整个流速范围内（从

纳升级到传统规格），可以保证 UHPLC 的能力，大大简化了分析的规模，而且不会影响到性能。

虽然蛋白质组学仍是纳升级液相色谱应用的主要焦点，但在生物制药和法医 / 毒理学应用方面亦

有很多潜力。如前所述，用户使用微型化液相色谱不但能提高灵敏度，而且更轻松地与质谱连接，

同时还大幅降低了样品和溶剂的消耗量。这不但补偿了延长的操作时间，还降低了操作成本。

切换到毛细管级液相色谱或微流速液相色谱意味着灵敏度和耐用性、通量和易用性之间达到完

美平衡。

所以重新回到标题中的问题：“液相色谱仪的微型化 ;为什么我们这样做呢？”，因为有很多裨益！

参考文献

1. Rieux, L.; Sneekes, E-J.; Swart, R. Nano LC: Principles, Evolution, and State-of-the-Art of the Technique. LCGC NA

2011, 29 (10), 926–934, [Online] http://www.chromatographyonline. com/lcgc/Column%3A+Innovations+in+HPLC/

Nano-LC-Principles-Evolution-and-State-of-the- Art-/ArticleStandard/Article/detail/745381 (accessed August 7,

2013).

2. Köcher, T.; Swart, R.; Mechtler, K. Anal. Chem. 2011, 83, 2699–2704.

3. Kiyonami, R.; Swart, R; Zabrouskov, V.; Huhmer, A. Increased Robustness and Throughput for Targeted Protein

Quantification Using Capillary Flow and a Conventional ESI Probe, Thermo Scientific Application Note 583, [Online]

http://www.thermoscientific.com/ecomm/servlet/techres ource?resourceId=102334&storeId=11152&from=sear

ch# (accessed August 7, 2013).

4. He, X.; Kozak, M.; Nimkar, S. Anal. Chem. 2012, 84 (18), 7643–7647.