Sa 安捷伦科技 HPLC色谱柱选择与维护保养  .

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1 Sa 安捷伦科技 HPLC色谱柱选择与维护保养

2 安捷伦HPLC色谱柱选择

3 分离模式的选择 用硅胶的正相模式 溶于正己烷 用键合相的正相模式 溶于甲醇或甲醇/水 溶于有机溶剂 或乙腈或乙腈/水 用键合相的反相模式
溶于四氢呋喃 分子量<2, 000 低分子凝胶渗透色谱 用键合相的反相模式 非离子化 溶于水 抑制电离反相键合相色谱 样品 离子化 离子对键合相的反相模式 用硅胶的正相模式 离子交换模式 溶于有机溶剂 凝胶渗透色谱 分子量>2, 000 凝胶过滤色谱 溶于水 大孔填料的离子交换模式 用大孔填料的反相模式

4 正相 &反相色谱 正相吸附色谱 反相分配色谱 极性:固定相 > 流动相 固定相 - 极性 流动相(己烷, 庚烷)- 非极性
极性物质后出峰 正相色谱 20% 反相分配色谱 极性:固定相 < 流动相 固定相 - 非极性 流动相(甲, 乙醇, 乙腈, THF, 二氯乙烷)- 极性 非极性物质后出峰 反相色谱 80%

5 柱填料基质 硅胶基质- pH 3~8 Al2O3 . nH2O - pH1~14 聚合物基质 - pH1~14 高或低pH下, 硅胶会溶解
化学修饰困难 孔结构复杂, 孔径不均匀导致柱效不够高, 有机溶剂可能导致聚合物基质溶涨而受损

6 C-18柱---性能影响因素 物理性质 硅胶纯度 色谱柱尺寸 颗粒形状 粒径 表面积 孔径 键合类型 碳覆盖率 封端 化学性质

7 C-18柱---性能影响因素 硅胶纯度 色谱柱尺寸 颗粒形状 粒径 表面积 孔径 色谱柱物理性质 • 填料硅胶的纯度与残留金属离子浓度
• 填料床的长度和内径 颗粒形状 • 球型或不规则型 粒径 • 平均颗粒直径, 通常3-10µm 表面积 • 颗粒外表面和内部孔表面的总和, 以m2/gram表示 孔径 • 颗粒的孔或腔的平均尺寸, 范围80-300Å

8 C-18柱---性能影响因素 键合类型 碳覆盖率 封端 色谱柱化学性质 • 单体键合 - 键合相分子与基体单点相连
• 聚合体键合 - 键合相分子与基体多点相连 碳覆盖率 • 与基体物质相连的键合相的量 封端 • 键合步骤之后, 用短链将裸露的硅羟基键合后封闭起来

9 C-18柱---性能影响因素 A类硅胶 B类硅胶 (高纯) Original ZORBAX SIL (1970s)
由于带负电荷的残留硅羟基和酸性表面上金属含量高 (硅羟基的pKa低), 导致碱性化合物发生拖尾 B类硅胶 (高纯) ZORBAX Rx-Sil (1987) 由于金属含量低, 硅羟基pKa高, 碱性化合物不发生拖尾 Zorbax Rx-SIL: 11种金属< 35 ppm (未检出其他杂质, <1ppm); % 纯度的二氧化硅

10 ZORBAX®生产工艺 安捷伦拥有此项溶胶-凝胶填料专利技术
StableBond Eclipse XDB Bonus-RP Extend 硅胶 + 脲 (pH=2) 甲醛CH2O 孔径 80A & 300A D O2 250oC 粒径 1.8 µm 3.5 µm 5.0 µm 7.0 µm ZORBAX Rx 多孔硅胶微球 安捷伦拥有此项溶胶-凝胶填料专利技术

11 C-18柱---性能影响因素 色谱柱尺寸 --- 对色谱分离的影响 • 短柱 (15-100mm) - 运行时间短, 柱压低
色谱柱尺寸 --- 对色谱分离的影响 • 短柱 (15-100mm) - 运行时间短, 柱压低 • 长柱 ( mm) - 分辨率高, 运行时间长 • 窄径柱 ( 2.1mm) - 检测器灵敏度高 • 宽径柱 (3-21.2mm) - 载样量高

12 C-18柱---性能影响因素 颗粒形状 --- 对色谱分离的影响 粒径 --- 对色谱分离的影响
颗粒形状 对色谱分离的影响 当使用黏度较大的流动相50:50=MeOH:H2O时, 球型颗粒可以降低柱压, 延长色谱柱寿命 球形 不规则形 粒径 对色谱分离的影响 较小的颗粒柱效较高, 但会引起柱压过高. 3.5µm粒径的常用于分离复杂的多组份样品, 而组份单一的样品多采用5µm的粒径 1.8µm µm µm µm µm

13 C-18柱---性能影响因素 表面积 --- 对色谱分离的影响 孔径 --- 对色谱分离的影响
表面积 对色谱分离的影响 高表面积对于多组份样品的分离具有较强的保留能力, 柱容量和分离度. 表面积低的填料通常能迅速达到平衡状态, 对于梯度淋洗尤为重要 孔径 对色谱分离的影响 大孔的填料颗粒可以延长溶质大分子在填料表面滞留的时间, 达到充分分离, 改善峰形 样品 MW  4, , 选择 80Å 的孔径 样品 MW > 4, , 选择 300Å 的孔径

14 大孔径填料适用于大分子分析 大孔径300Å 全多孔色谱柱 Poroshell 色谱柱 可用于分离蛋白质和多肽
分子量虽小但hydrodynamic volume(水动力学体积)较大的分子 Poroshell 色谱柱 高流速下, 高柱效分析大分子的蛋白质和多肽 色谱柱填料孔径必须适合待测物分子自由进出填料孔, 与孔内表面的键合相进行分离分配 Thus far we have covered 硅胶和键合相s 和their impact on 色谱峰峰形, 和this has focused mainly on improving the 色谱峰峰形 of basic analytes. These would also be characterized as small molecules thus far. But good 色谱峰峰形 is equally important with larger molecules – such as 蛋白 和peptides. Good 色谱峰峰形 with these analytes is more than just a lack of 拖尾峰ing. It is also a sharp efficient peak. This can be even more important than 拖尾峰ing because most 蛋白质和多肽 are separated by 梯度洗脱 methods 和拖尾峰ing is typically not as evident in 梯度洗脱 separations. To analyze these types of molecules 全多孔wide pore 300Å 色谱柱 can be selected to achieve good 色谱峰峰形 – sharp efficient peaks. This does not just apply to 蛋白质和多肽, but lower molecular weight molecules with large hydrodynamic volumes – those that take up a large space in solution. A third 色谱柱 choice for good 色谱峰峰形 with 蛋白质和多肽 和other 大分子is a new 色谱柱 type called Poroshell. This is exactly what it sounds like. A 多孔外壳, with the 300A 孔径, surrounds a solid core. This is ideal for separations of 大分子at higher 流速s. So let’s start looking at how 和why these 色谱柱 types impact 色谱峰峰形.

15 300Å 孔径可改善蛋白质和多肽峰形 孔径, 分子量对峰宽的影响(梯度分离) 300SB-C18 (300Å) SB-C18 (80Å)
选择合适的 填料孔径分析 大分子可获得 最佳峰形 0.20 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 300SB-C18 (300Å) SB-C18 (80Å) PW 1/2 Leu Enkephalin M.W. = 556 Angiotensin II(血管紧张素 II) M.W. = 1046 M.W. = 3, Insulin B M.W. = 12, Cyt C Rnase(核糖核酸酶) M.W. = 13, Lysozyme(溶菌酶) M.W. = 13, 孔径, 分子量对峰宽的影响(梯度分离) This slide shows the effect of 孔径 和molecular size on peak width for 梯度洗脱 separations. Early in the talk we mentioned that peak width at half height was a good indicator of 色谱峰峰形 because very broad peaks usually indicate 二次作用 or a peak that. It can also indicate a compound is not freely diffusing in 和out of the 色谱柱 pores. This could be because the pore is not big enough for free diffusion. This graph is designed to help us determine what molecular weight is too big for a standard 80Å 孔径 色谱柱 和when a 300Å 孔径 色谱柱 is needed. We measure this by the change in the peak width at half height. We can see that this occurs at a molecular weight of greater than So for these 大分子it is important to choose a 300Å 孔径 色谱柱, 和a 全多孔wide pore 色谱柱 is a standard first choice.

16 300Å 孔径可改善大分子的峰形 SB-C3 (80Å) 300SB-C3 (300Å) 溶液中的分子尺寸决定适宜的色谱柱孔径
色谱柱: 4.6 x 150 mm, mm 流动相: 60% MeOH: 40% 0.1% 三氟乙酸 流速: 0.75 mL/min 温度: RT 检测器: UV 282 nm 分子量虽小但 hydrodynamic volume (水动力学体积)较大的分子 SB-C3 (80Å) 300SB-C3 (300Å) Pw 1/2 = 0.442 Pw 1/2 =0.125 O N HO CHO H CH 3 OCH 2 C Time (min) Tylosin(泰乐菌素) MW. 926 Choosing wide pore 色谱柱 for efficient peaks is also important for molecules that have a large hydrodynamic volume. The molecule shown here is Tylosin(泰乐菌素), with a molecular weight of only You would not normally think that this molecule needs a large 孔径 色谱柱, but if we look at the difference in peak widths at half height on the 80 和300Å 孔径 色谱柱 we can see that it is substantial. The 80Å 孔径 色谱柱 gives a peak width at half height of where as the 300SB-C3 gives a peak width at half height of only This is the peak width at half height that I would expect for the retention time of this peak. If we just used 柱效 or 拖尾因子 of an indictor of 色谱峰峰形 problems we would know this is a gaussian peak with low 柱效. The peak width at half height is our strongest indicator of what the problem is with this peak 和here it is telling us that the 孔径 is not big enough for this analyte. The narrow peak width indicates unrestricted access to the pores. Once the proper 柱效 和peak width is achieved greater resolution can be obtained if there were additional compounds present. 溶液中的分子尺寸决定适宜的色谱柱孔径 窄的峰宽表明待测分子进入填料孔没有受到阻碍

17 C-18柱---性能影响因素 键合类型 --- 对色谱分离的影响 单齿键合： 提高传质速率, 加快色谱柱平衡 双齿键合：
键合类型 对色谱分离的影响 单齿键合： 提高传质速率, 加快色谱柱平衡 双齿键合： 增加色谱柱稳定性, 增加色谱柱的载样量 CH O Si 3

18 C-18柱---性能影响因素 碳覆盖率--- 对色谱分离的影响 封端 --- 对色谱分离的影响 高碳覆盖率：提高分辨率, 分析时间长
碳覆盖率--- 对色谱分离的影响 高碳覆盖率：提高分辨率, 分析时间长 低碳覆盖率：缩短运行时间 封端 对色谱分离的影响 封端：减轻待测组份与硅胶表面残留的酸性硅羟基反应而引起的色谱峰拖尾现象 对于极性样品, 未封端与经过封端处理的色谱柱在选择性上有明显差异

19 传统键合及封端技术 + + 封端 固定相键合 四甲基硅烷 二甲基硅烷 CH CH O Si R O Si R OH CH CH CH OH
3 CH 3 O Si R O Si R OH CH CH 3 CH OH 3 OH CH 3 OH CH + 3 + 3 Cl Si R Cl Si CH CH OH CH 3 O Si OH 3 3 CH CH CH 3 3 OH O Si R 3 CH 3 R = C8, C18, etc . ( TMS) CH O Si R 3 封端 四甲基硅烷 固定相键合 二甲基硅烷

20 安捷伦专利的空间位阻保护 Zorbax StableBond 普通硅胶柱低 pH <2 空间位阻保护---抗水解能力
取代基：五个 旁侧基团: 异丙基 键合相 流失, 保留减弱 另一个裸露的硅羟基 , 潜在的拖尾因素 Zorbax StableBond 空间位阻保护---抗水解能力 普通硅胶柱低 pH <2 不稳定, 发生水解

21 液相柱---保留值与pH的关系 pH变化值 0.1 RS变化值1.6 色谱柱：Zorbax StableBond C18
4.6 X 250mm, 5um 部件号： 流动相：27%甲醇：73%磷酸 pH 2.5 & 2.6 温度：500C 流速： 1.0mL/min. pH变化值 0.1 RS变化值1.6

22 液相柱---保留值与pH的关系 pH 7.00 pH 7.25 0.25 个pH
色谱柱: ZORBAX Eclipse XDB-C8 4.6 x 75 mm, µm 流动相: 44% 25 mM磷酸, pH 7.00 : 56% 甲醇 流速: 1.0 mL/min 温度: 25°C 检测: UV 250 nm 1 2 3 4 6 7 5 Time (min) pH 7.00 pH 7.25 0.25 个pH 样品: ketoprofen ethyl paraben Hydrocortisone fenoprofen propyl paraben Propranolol ibuprofen 1.      测试f或pH方法可靠性 This sl内径e shows analgesic/neutral mixture again和highlights 变化 in 选择性 that can occur f或ionized 待测组份. On a pH scale this is a not a small 变化 at all. 差异 such as 0.25 或0.50 pH units are used when using computer模拟to find most robust pH f或a分离. Frequently, pH 差异 as little as pH units can show marked 变化in 色谱峰保留和流出顺序. To 评估pH方法可靠性 you should 对比分离at desired 或优化pH  units. 可电离的组份的分离可能会随pH变化发生显著变化 —甚至很小的 0.05–0.25 个pH变化单位.

23 液相柱---保留值与pH的关系 pH变化： 0.2个pH(7.0~7.2) 保留时间改变： 1.2分钟 (13.6~14.8分钟)
改变不明显 保留时间随pH改变 不明显(酸性环境中)

24 液相柱---保留值与pH的关系 保留时间 酸性组份 中性组份 碱性组份 方法稳定区 方法波动区(pKa+1.5) 方法稳定区

25 液相柱---保留值与pH的关系 Bonus-RP pH 2~9 Extend C18 pH 2~11.5
Eclipse XDB C18 pH 2~9 StableBond C18 pH 1~8

26 复杂组分的流动相pH选择技巧 Extend-C18 Extend-C18 pH 7 高pH下, 碱性成分的保留增加 pH 11
4 4 2, 3 3 1 5 1 5 2 6 6 1. Maleate 马来酸盐 2. Scopolamine 东莨菪碱 pKa 7.6 3. PseudoEphedrine 假麻黄碱 pKa 9.8 4. Doxylamine 抗敏安(多西拉敏) pKa 9.2 5. Chlorpheniramine 氯苯吡胺pKa 9.1 6. Triprol内径ine 苯丙烯啶pKa 6.5 7. Diphenhydramine 苯海拉明 pKa 9.0 Time (min) Time (min) We took a 样品 of basic cough 和cold components 和analyzed them on the Extend-C18 色谱柱 at m内径 和高pH下. In general you want to work at least 1 pH unit above the pKa of a basic analyte to be working with it as a fully non-charged analyte – preferably 1.5 pH units. Notice that most of these components have pKa’s of 9 or higher – one is 7.6 和one is pH Therefore, only one of these compounds can be analyzed uncharged in the typical pH 范围 of most 色谱柱. So 高pH下 is a good choice for this 样品. At m内径 pH – pH 7 - some 拖尾峰ing is ev内径ent on the Chlorpheniramine 氯苯吡胺 (氯苯吡胺) 和Diphenhydramine (苯海拉明). But at 高pH下- pH 11 (more than 1 pH unit above the highest pKa) the 色谱峰峰形 of all of the components is excellent. Comparing the same organic strength 流动相s also shows us that more retention results. This is another advantage of working at 高pH下 with basic compounds. Notice that this additional retention allows more resolution between two of the peak pairs. This separation is 内径eal at 高pH下 和the high organic will help increase the 色谱柱 lifetime even more. These are several 键合相 types, 封端, with 极性embedded groups, 和ones for use at 高pH下 that can be used to improve 色谱峰峰形. These are not the only approaches to 键合相s – but these three are key choices for improving the 色谱峰峰形 of 碱性组分和they all take a slightly different approach. This means that if one solution is not right for your analysis another most likely will be. This is an important secret of getting good 色谱峰峰形 – there is more than one approach 和the right one for your analysis is most likely available. You many not always know the best approach at first but anything that you learn makes finding the answer easier on the next try. 待测物pKa+1.5以外 的流动相pH 可保证方法的稳定性 色谱柱: ZORBAX Extend-C18 4.6 x 150 mm, mm 流动相: 30% 缓冲液: 70% MeOH pH 7 缓冲液20 mM Na2HPO4 pH 11缓冲液 20 mM TEA 流速: 1.0 mL/min 温度: RT 检测器: UV 254 nm

27 Zorbax色谱柱发展史 1973 ZORBAX SIL 发明专用于HPLC分离的球状、小颗粒硅胶(专利)
1977 ZORBAX ODS 单官能团, 单层键合相发明者之一 1978 ZORBAX column 首先推出色谱柱的质量信誉保证 1982 ZORBAX GF-250 改善了蛋白质尺寸分离的稳定性 1987 ZORBAX Rx-SIL 推出适合碱性化合物的HPLC分离的全新硅溶胶技术(专利) 1990 ZORBAX StableBond 增强了低pH条件下键合相的稳定性(专利) 1996 ZORBAX Eclipse XDB 双封端技术提高了中等pH条件下键合相的稳定性(专利) 1997 Zorbax LC/MS column 15和30 mm(3.5um)快速, 高分辨率, 高通量LC/MS色谱柱 1998 ZORBAX Bonus-RP 极性键合技术使碱性化合物的分离得到改善(专利) 1999 ZORBAX CombiHT 专为小规模组合化学样品制备设计的高通量色谱柱 1999 ZORBAX Eclipse dsDNA 用于分析双链DNA的多孔硅胶柱 1999 ZORBAX Carbohydrate 分析单, 双, 三糖和糖醇的专用柱 1999 Zorbax Extend C18 独特宽pH跨度, 高pH下使用的硅胶基质色谱柱(专利) 2001 Zorbax SB-aq 可在低pH下(1~8)使用100%水相的填料(专利) 2002 Zorbax Poroshell 300SB 蛋白质高速, 高效反相分离新填料 2002 ZORBAX 3.0mm 内径. 3-mm 柱径溶剂节省柱 2003 ZORBAX sub 2 micron 1.8um粒径的高通量, 超高速色谱柱 (5月1日上市) 2003 ZORBAX PrepHT 高通量全系列制备柱---分析柱向制备柱直接放大 (6月1日上市)

28 Silica-based 色谱柱 Packing with Alkyl/am内径e Stationary Phase
StableBond, pH 1-8 1. 使用空间位阻大的硅烷 2. 未-封端 Eclipse XDB, pH 2-9 1. 致密键合的二甲基烷基硅烷 2. 专利技术双 - 封端 Bonus-RP, pH 2-9 1. 极性烷基固定相 2. 使用空间位阻大的硅烷 3. 三封端 Extend-C18, pH 1. 独特的双齿键合结构 2. 双封端 低pH下, 分析酸性, 碱性和中性组分, 峰形优异, 柱寿命长 中等pH下, 分析酸性, 碱性和中性组分, 尤其碱性组分峰形优异, 柱寿命长 中等pH下, 分析酸性, 碱性和中性组分, 可用于100%全水相, 尤其极性组分峰形优异, 柱寿命长 中~高pH下, 分析酸性, 碱性和中性组分, , 尤其碱性组分峰形优异, 柱寿命长 Si O Silica Support C18 NH Silica-based 色谱柱 Packing with Alkyl/am内径e Stationary Phase R OH CH 3 1 R1 1 1 PG R 1 极性官能团

29 Zorbax 产品系列 反相色谱柱 StableBond Eclipse XDB Extend 低pH 中pH 高pH Bonus-RP
空间位阻保护 Eclipse XDB 双封端 Extend 双齿键合+双封端 Bonus-RP 空间位阻保护 + 三封端 80 A pH: 2~9 SB-C18 80 A & 300 A pH: 1~8 SB-CN 80 A & 300 A pH: 1~8 Eclipse XDB-C18 80 A pH: 2~9 Extend-C18 80 A & 300 A pH: 2~11.5 可使用 100%水相 SB-C8 80 A & 300 A pH: 1~8 SB-C3 80 A & 300 A pH: 1~8 Eclipse XDB-C8 80 A pH: 2~9 SB-AQ 空间位阻保护 80 A pH: 1~8 SB-苯基 80 A pH: 1~8 Poroshell 300SB-C18 实心核多孔壳 pH: 1~8 Eclipse XDB-苯基 80 A pH: 2~9

30 安捷伦HPLC色谱柱维护保养

31 Zorbax色谱柱=高柱效 +长寿命 柱效 进样次数 #3 #4 1 6635 6138 100 6857 6372
进样次数 #3 #4 色谱柱: ZORBAX SB-C18 4.6 x 30 mm, 1.8um 流动相: 60% 甲醇: 40 水 流速: 1mL/min 温度: 室温 检测: UV 254 nm 2 Norm. 50 40 样品 尿嘧啶 苯酚 4-Cl-硝基苯 甲苯 1 30 3 4 Another benefit of 快速分离高通量（RRHT）色谱柱 is that they have a long useable life时间. Our study was continued beyond 什么是shown here -- up to more than 2500 injections, but at that point we were only at 95% of the或iginal 柱效和chose to cut off the reported data greater than 2000 injections because 2000 injections is the normal point at which many 高通量 实验室 choose to change色谱柱 for a normal life时间, 和so we can demonstrate that there is absolutely no difference up to that point in时间. This is a good life时间 – the variation in the Toluene peak is just due to the volatility of toluene. During this study, more than 50 injections per vial were made, which is probably pushing the limits. The other peaks are all dead on. Another 关键observation from this data is that retention时间s和tailing factors remain constant over时间. 20 10 0.5 1 1.5 2 min

32 色谱柱柱内体积和平衡时间 尺寸 体积 流速 (mm) (Vm) 1.0 mL/min
4.6 x mL 5 min 4.6 x mL 3 min 4.6 x mL 1.5 min 4.6 x mL 15 min 2.1 x mL 5 min 60 sec 2.1 x mL 3 min 36 sec 2.1 x mL 1.5 min 18 sec 色谱柱 柱内 平衡时间 尺寸 体积 流速 (mm) (Vm) mL/min (mm) (Vm) mL/min mL/min This slide shows just why we say that梯度 equilibration is not a limitation when using 短色谱柱. Most色谱柱 require “ ten时间s their色谱柱 volume” to obtain reproducible retention during the梯度 run. A 标准4.6 x 150 mm色谱柱 has an internal volume of 1.54 mL, which means that 15 mL of溶剂are requi红色 to equilibrate that色谱柱 –和at a 流速 of 1.0 mL/min. it takes 15 minutes to accomplish this task. For the 短 4.6 mm i.d.色谱柱 this is 短en considerably from 5 – 1.5 minutes.和for the 2.1 mm i.d.色谱柱 operated at 1.0 mL/min., equilibration时间 is 缩短d to a 60 to 18 seconds….it takes more时间 to ready the next injection. 单次样品运行时间 = 分析时间 + 色谱柱平衡时间 色谱柱平衡时间 = 10倍柱内体积 ÷ 流速

33 清洗液相色谱柱 冲洗色谱柱---用比流动相更强的溶剂 反相色谱柱 用以下溶剂至少各25mL冲洗色谱柱(分析柱) 不含缓冲盐的流动相
100%甲醇 100%乙腈 75%乙腈+25%异丙醇 100%异丙醇 100%二氯甲烷 * 100%己烷* *若使用己烷或二氯甲烷冲洗色谱柱, 则在重新使用反相 流动相以前, 必须用异丙醇冲洗色谱柱 !!! 正相色谱柱 用以下溶剂至少各50mL冲洗色谱柱(分析柱) 50%甲醇+50%三氯甲烷 100%乙酸乙酯 Flush the column with stronger solvents than your mobile phase in order to clean it. If you are cleaning a reversed-phase column then some appropriate solvent choices are listed here. You should use at least 25 mL of each solvent for a standard 4.6 mm 内径 analytical column. Smaller 体积s can be used with smaller 内径 columns. The first step is to wash the column with the mobile phase without buffer salts. This will wash out the salts and eliminate any possibility of precipitation before going on to stronger solvents. Methanol and acetonitrile are the most common wash solvents and the first choice would be the one in your mobile phase. Then I like to use a mix of ACN and IPA. It will eliminate a lot of contaminants without the pressure generated by 100% isopropanol. Some people will jump right to 100% IPA because it is stronger. Stronger still are methylene chlor内径e and hexane. If you use either of these solvents it is important to note that to get back to your mobile phase you must pass through IPA because it is miscible with these solvents and acetonitrile and methanol. Note that we do not encourage washing the column with 100% water. This does not help remove strongly retained compounds in reversed-phase HPLC and can lead to further column contamination. Buffer salts will not precipitate if you wash with your mobile phase aqueous organic composition without the salts. Cleaning a column used for normal phase chromatography requires choosing solvents that are stronger from the normal-phase perspective. That is choose solvents that are more and more polar. Some good solvent choices are a mix of methanol and chloroform and then ethyl acetate. It is also possible to use small amounts of ac内径 for instance to eliminate strongly retained material. But this needs to be done with caution. Silica columns can take a long time to re-equilibrate with your mobile phase after washing with a water containing solvent. This sl内径e shows you how to change a frit. Changing a frit is a last resort procedure because you risk destroying the column efficiency. But this shows you how to do it. The first thing is to have the column held upright in some type of clamp. Do not touch the column because body heat can warm up the solvents in the column and cause packing material to extrude and be lost when the frit is changed. Lost packing material means lost efficiency. Then open up the column and take the frit off the top of the column bed or out of the end fitting. Do not allow the column bed to dry out while you are doing this. Keep if wet with acetonitrile or methanol. Then put in the new frit of the same type and dimensions as the one you removed. Check with column manufacturer if you are not certain if you have the correct frit. Finally reassemble the end fitting and do not overtighten the fitting. This is not a difficult procedure, but it should only be done at the inlet frit. ZORBAX columns only have a replaceable inlet frit, and not an outlet frit. The outlet frit is pressed in for a more efficient column packed bed. Any time this process does not go smoothly the column may not be useable after the frit change. So always be prepared with a backup column.

34 连接管线与接头 <0.12mm内径管线用于毛细管HPLC中<2.1内径色谱柱
10 mL 柱外体积 Time (min) 4 3 2 1 4 3 2 1 Time (min) 样品: 1. 苯丙胺酸 2. 5-苯基-3, 6-二氧-2-哌嗪乙酸 3. Asp-Phe 4. 天冬甜素 50 mL 柱外体积（管线） 色谱柱: StableBond SB-C18, 4.6 x 30 mm, 3.5 mm 流动相: 85% H2O with 0.1% 三氟乙酸 : 15% ACN 流速: 1.0 mL/min 温度: 35°C <0.12mm内径管线用于毛细管HPLC中<2.1内径色谱柱 0.17mm内径管线用于分析型HPLC中2.1~4.6内径色谱柱 0.25mm内径管线用于半制备型HPLC中9.4内径色谱柱 0. 5mm内径管线用于制备型HPLC中21.2内径色谱柱

35 HPLC常用的色谱柱接头 Swagelok 安捷伦1100液相 Waters Parker Rheodyne Valco Uptight
0.09 in. = 2.33 mm Swagelok 安捷伦1100液相 Parker Valco Uptight 0.08 in. = 2.07 mm 0.13 in. = 3.37mm 0.17 in. = 4.40mm Waters Rheodyne To continue with the concept of matched fittings, this slide shows various column connectors used in HPLC. Notice that for each vendor, the distance from the end of the tubing to where the ferrules are "seated" or "swaged" is different? If the distance is too great for the end fitting of the column, the ferrule will not seal with the column and the fitting will leak. If you then tighten down on the fitting, the tubing will puncture the column frit. If the distance is too short, you can seal the ferrule and not have a leak, but there will be a large extra column volume for band broadening to occur. Notice how the ferrules are shaped differently as well? Some are designed to be used with columns and some are specific to injectors or detectors or pumps. DO NOT INTERCHANGE THESE! They may become a permanent fixture, or imbedded in your column or equipment. The last point is, once you seat the ferrule or swage it into place, that fitting is the right size for only that column type. Each column end fitting or holder from one vendor is not machined the same as another vendor. Thread number and depth vary. A cartridge system with the same end holders for all cartridges prevents you from needing to have multiple dedicated end fittings for multiple columns. Many users are changing to universal end fittings which are plastic or PEEK, yet seal at high pressures, are adjustable, and re-usable. There is little chance of mismatch. Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch. LC/GC Magazine 6:788 (1988)

36 HPLC常用的色谱柱接头 如果伸出的管线长度过长，可能漏液 螺母坡度不匹配，密封性差 死体积区 如果伸出的管线长度不够，可能产生死体积

37 活塞杆与密封圈 *己烷对Vespel聚酰亚胺有溶涨作用 蓝宝石活塞杆 1/包装 部件号：5063-6586 活塞杆密封圈
蓝宝石活塞杆 1/包装 部件号： 活塞杆密封圈 （Vespel聚酰亚胺用于反相） 2/包装 部件号： *己烷对Vespel聚酰亚胺有溶涨作用 （聚乙烯用于正相） 2/包装 部件号：

38 活塞杆与密封圈 色谱柱: Bonus - RP 4.6 x 75 mm, 3.5 mm 流动相: 30% H2O : 70% MeOH 流速: 1.0mL/min 温度：室温 检测器: UV 254nm Time (min) 2 1 3 塔板数 USP拖尾因子 (5%) 塔板数 USP拖尾因子 (5%) In this example if we look at the injection on the left we are disappointed in the performance of the column. It’s not bad, but it is not good. What stood out most to us was the low plate counts on all the peaks, though there was more peak tailing than we expected on each peak. We knew that this column should have given more plates. We then had to figure out what the problem was. Since it was a new column and we had no reason to expect column failure, we tried it on another HPLC system and got much higher plates. So we tried to determine which component of our system was not working properly and we d内径 it by substituting in one piece at a time. We put in a new injector and got much better results. Therefore we d内径 routine maintenance of our injector and replaced the seals. Then we got much better results. This is another problem that appears to be a column problem, but comes from another part of the system. Replacing suspect pieces one at a time is the best way to determine what part is causing the problem. Injector seal failure causes the loss of efficiency and tailing because when these parts are not working there is unswept 体积 in the system causing these problems. 更换前 更换后 样品: 尿嘧啶 苯酚 N, N-二甲基苯胺

39 检测器用灯

40 检测器用灯

41 检测器用灯

42 DAD的氘灯替代VWD的氘灯 1. DAD的氘灯替代VWD的氘灯 关闭灯电源, 冷却5min以上
将lamp设定的VWD configuration的灯类型改为 2. 灯能量测试 以水为流动相 灯预热10min以上 在化学工作站Diagnosis状态下进行波长校准, 将紫外灯的使用时间归零

43 DAD故障诊断与维护 要进行DAD故障诊断, 单击DAD图标, 然后选择Show Module Test选项, 进入DAD测试选项画面
DAD Self-Test DAD Intensity Test DAD Cell Test 常做的DAD维护： 更换灯 清洗或更换流动池窗

44 Diagnosis界面故障原因分析 诊断过程故障分析
样品分析过程中故障分析 在Diagnosis画面中为用户提供了在诊断过程或样品分析过程中出现的问题以及可能的原因 可能原因框 故障选择框

45 样品溶剂溶解强度 A. 样品溶剂 100% 乙腈 B. 样品溶剂 流动相 样品溶剂溶解能力强于流动相导致色谱峰峰形问题：裂分峰或宽峰
色谱柱: ZORBAX StableBond SB-C8, 4.6 x 150 mm, 5 mm 流动相: 82% H2O : 18% ACN 进样体积: 30 mL A. 样品溶剂 100% 乙腈 Time (min) 1 2 B. 样品溶剂 流动相 样品: 1. Caffeine (咖啡因) 2. Salicylamide (水杨酰胺) The 样品溶剂 must be considered whenever you see poor 色谱峰峰形, especially if you have compounds that elute early in the run. It is possible to see 峰形前伸ing from a strong 样品溶剂. The 峰形前伸ing can look just like a fronting peak or it could possibly be a partially split peak like the one we see here, or I have seen it split more into two unresolved peaks. In this example the 样品溶剂 is 30 uL of 乙腈 in the A chromatogram. The 流动相 is only 18% 乙腈, so this is a lot stronger. The result is a split peak on the first peak. Notice the second peak as well. That peak is much broader 和tails a little. This would probably just be noticed as less 柱效 than would be expected. If the 样品溶剂 is the 流动相 the result is two very sharp peaks with excellent 色谱峰峰形. The 进样体积 here is 30 uL, but be aware that this can occur with even lower 进样体积s depending on elution times 和流动相 composition. These two things, 样品 solvent strength 和corresponding 进样体积 as well as 样品 load on the 色谱柱 are the two primary factors of 色谱峰峰形 problems. 样品 solubility, though not specifically shown here affects both of these 和therefore 色谱峰峰形. Start by dissolving your 样品 in whatever solvent it is most soluble 和then dilute to match your 流动相. Do this to obtain good solubility 和limit 色谱峰峰形 problems. 样品溶剂溶解能力强于流动相导致色谱峰峰形问题：裂分峰或宽峰

46 含缓冲液的流动相可改善保留, 分离和色谱峰峰形.
中性分析时为何要使用缓冲液？ 色谱柱: ZORBAX Eclipse XDB-C8, 4.6 x 75 mm, 3.5 µm 流动相: 44% A : 56% 甲醇 流速: 1.0 mL/min 温度: 25°C 检测: UV 250 nm 样品: 1. ketoprofen 2. ethyl paraben 3. hydrocortisone 4. fenoprofen 5. propyl paraben 6. propranolol 7. ibuprofen A = pH 7.0水 A = pH 水+25 mM 磷酸缓冲液 1 2 3 4 5 Time (min) 6 7 1, 4, 6, 7 1.      Why Use 含缓冲液的流动相? This sl内径e shows分离 of a mixture of ac内径ic和neutral 待测组份 carried out at pH These are non-内径eal conditions f或analyzing these ac内径s, but were chosen f或demonstration purposes. The分离 on left shows the分离 using pH-adjusted水 in 流动相. This was prepared with a minimum of phosphoric ac内径和钾hydrox内径e solution. pH of deionized和organic-free水 can 改变a 批号 on a daily basis, as a function of source 或br和of purifier, 时间 of year, amount of dissolved carbon diox内径e, 等, 等 All ac内径s elute very early as they are charged和the硅胶surface is negatively charged at this pH. last two 组份 色谱峰 tail more than is desirable at this pH. This 色谱峰峰形 can be improved by 增加 缓冲液浓度 to mM 或by adding 10 mM triethylamine to 流动相. 含缓冲液的流动相可改善保留, 分离和色谱峰峰形.

47 增加缓冲液浓度减少拖尾 10 mM 磷酸盐 pH 7.0 25 mM 磷酸盐 pH 7.0
色谱柱: ZORBAX Eclipse XDB-C8 4.6 x 150 mm, 5 mm 流动相: 40% 磷酸盐缓冲液 60% ACN 流速: 1.5 mL/min. 温度: 40°C USP Tf 1.41 1.50 1.33 1.39 1.36 1.00 USP Tf 1.62 1.65 1.63 1.77 1.83 1.12 Time (min) 1 2 3 4 5 6 3 4 5 6 样品: Tricyclic Antidepressants （三环抗抑郁剂） 1. Desipramine（脱甲丙咪嗪） 2. Nortriptyline（去甲替林） 3. Doxepin（多塞平） 4. Imipramine（丙咪嗪） 5. Amitriptyline（阿米替林） 6. TrImipramine（三甲丙咪嗪） Not only is it important to use 缓冲液ed 流动相s, optimizing the 缓冲液 concentration can improve 色谱峰峰形. This is especially important at mid pH where the 缓冲液 can mask 硅羟基interactions between the analytes 和residual 硅羟基. The example here shows a group of basic compounds, antidepressants, at pH 7 with both 10 mM 和25 mM 磷酸盐缓冲液trength. The 拖尾因子s of these compounds are all lower at 25 mM 磷酸盐缓冲液than at 10 mM. The increased 缓冲液 strength 降低the effect of interactions with the charged 硅羟基. We typically recommend a 缓冲液 strength of 25 mM to start. This will give you more consistent control of the 色谱柱 硅羟基 和the 离子化 of your analytes resulting in a more reproducible method. 在中等pH下, 离子强度较高, 能有效掩蔽硅羟基的二次作用减少拖尾

48 TEA对碱性成分峰形的影响 pH 7 10 mM TEA No TEA
色谱柱: ZORBAX Eclipse XDB-C8, 4.6 x 150 mm, 5 mm 流动相: 85% 25 mM Na2HPO4 : 15% ACN 流速: 1.0 mL/min. 温度: 35°C pH 7 10 mM TEA Time (min) 0.0 2.5 5.0 3 5 4 2 1 1 No TEA USP TF 5% 样品: 1. Phenylpropanolamine （去甲麻黄碱） 2. Ephedrine（麻黄素） 3. Amphetamine（苯丙胺） 4. MethAmphetamine（脱氧麻黄碱） 5. Phenteramine （苯丁胺） USP TF 5% 3 2 4 5 One of the last things you can do to improve 色谱峰峰形 is add additional 流动相改性剂. These are things like triethylamine – TEA – added to interact with charged 硅羟基 so that your basic analytes will not ion exchange, causing poor 色谱峰峰形. That is what we have done here. We took the same basic amines you saw earlier 和starting at pH 7 where the 拖尾因子 of methAmphetamine（苯丙胺） was 2.35, we added 10 mM TEA to the 流动相 和kept the pH at 7. The result is improved 色谱峰峰形. What is interesting about these results is that the 拖尾因子 is improved by the same amount, as it was when we lowered the pH to 3. So working at low pH can mean a simpler 流动相, with less likelihood that any 流动相 additives will be necessary for good 色谱峰峰形. We should mention that we are using the Eclipse XDB 双封端 色谱柱, but there will be some 硅羟基 on every 色谱柱, especially at pH 7, that will ionize 和are available for the 离子交换 interactions that cause poor 色谱峰峰形. Though additional 流动相改性剂 are rarely necessary, some very difficult 样品s could require them for good 色谱峰峰形. Time (min)

49 竞争酸对酸性组分的峰形的影响 A: pH 3 5 mM NaH2PO4 pH 3 5 mM NaH2PO4 1% 乙酸 pH 2.5
0.1% 三氟乙酸 色谱柱: ZORBAX StableBond SB-C18 4.6 x 150 mm, 5 mm 流动相: 40% A: 60% ACN 流速: 1.0 mL/min. 温度: 室温 样品: Ibuprofen（异丁苯丙酸） pKa 4.4 Acidic compounds sometimes also have 色谱峰峰形 problems, especially with very low ionic strength 流动相s. Just as with basic compounds, where you add a competing base to reduce 拖尾峰ing, with acids you can add a competing acid. The competing acid generally interrupts hydrogen-bonding type of interactions. In this case we start with a very weak 磷酸盐缓冲液– only 5 mM at pH 3 - 和get a 拖尾因子 of 1.8 for the carboxylic acid containing compound. If we add a competing acid – 1% 乙酸 – the 色谱峰峰形 improves with a perfect 拖尾因子. But 1% 乙酸 is a lot. So we tried another competing acid – 三氟乙酸 – Trifluoro乙酸 和used only 0.1%. This gives us a very good result with a much simpler 流动相. 三氟乙酸 can be used at a much lower concentration 和that is often preferred. There will be less likelihood of any unnecessary background absorbance with the 三氟乙酸, than with 1% 乙酸. (If they remember or you want to point out from earlier on – good 色谱峰峰形 can be obtained by raising the 缓冲液 strength as well.) Tf = 1.8 Tf = 1.0 Tf = 1.09 乙酸和三氟乙酸均能作为竞争酸填加至流动相中 三氟乙酸由于所需浓度较低, 可作为首选

50 预混合（重量比混合）: Premixed (w/w)
色谱柱技术 – EN 流动相对色谱的影响(TE40) 液相色谱柱: ZORBAX Eclipse XDB-C8 色谱柱尺寸: 4.6 x 50 mm 3.5µm 部件号: 改变流动相配比方式, 可增加柱效 （提高灵敏度） UV: 254 nm 流速: 1 mL/ min. 室温 尿嘧啶 尼帕尔金丁酯 萘 二丙基邻苯二甲酸酯 苊 Uracil Butylparaben Naphthalene Dipropylphthalate Acenaphthene 刻度混合: Dial-a-Mix A: 水 B: 甲醇, 用泵抽取50% B 体积比混合: Vol/Vol 250 mL水 mL甲醇, 用泵抽取100%混合液 定容混合: Mix-to-Mark 250 mL甲醇, 用水定容至500 mL, 用泵抽取100%混合液 预混合（重量比混合）: Premixed (w/w) 200 g甲醇 g水, 用泵抽取100%混合液

51 液相分析-小诀窍 提高柱温，可增加柱效（提高灵敏度） 减小柱内径，可增加柱效（提高灵敏度） 缩短检测器响应时间，可增加柱效（提高灵敏度）
减小柱外体积，可增加柱效（提高灵敏度） 使用超纯硅胶，可增加柱效（提高灵敏度） 使用高能量氘灯，可增加柱效（提高灵敏度） 使用中孔透光氘灯，可增加柱效（提高灵敏度） 改变流动相pH ，可增加柱效（提高灵敏度） 改变有机相% ，可增加柱效（提高灵敏度） 改变键合相 ，可增加柱效（提高灵敏度） 改变有机添加剂 ，可增加柱效（提高灵敏度） 改变流动相配比方式 ，可增加柱效（提高灵敏度）

52 提高柱温，可增加柱效（提高灵敏度） 液相色谱柱: ZORBAX StableBond-C18 色谱柱尺寸: 4.6 x 250 mm 5µm
生化 –多肽 EN 升温对于反相 HPLC分离强疏水肽的影响 (B27) Polypeptide 液相色谱柱: ZORBAX StableBond-C18 色谱柱尺寸: 4.6 x 250 mm 5µm 部件号: 室温 300C 400C 500C 600C 700C 流动相: A: 含0.05%三氟乙酸的水 B: 含0.05%三氟乙酸的乙腈 2% B / min 进样量: 100µL (50µg的6M脲/5%乙酸) UV: 210 nm 流速: 1 mL/min 室温 提高柱温，可增加柱效（提高灵敏度）

53 提高柱温，可增加柱效（提高灵敏度） 目录P625 液相色谱柱: Zorbax StableBond-C18
环保 液相色谱柱: Zorbax StableBond-C18 色谱柱尺寸: 4.6 x 150 mm, 5μm 部件号: 除草剂: 柱温的影响 Herbicides: Effect of Column Temperature 40℃ 1. 西玛津 2. 敌草隆 3. 扑灭津 4. 扑灭净 5. 异丙甲草胺 样品: 除草剂 流动相: 52/ M乙酸钠/甲醇, pH 6 流速: mL/min 温度: ℃, 90℃ 检测: UV 90℃ 1. Simazine 2. Diuron 3. Propazine 4. Prometryn 5. Metolachlor 提高柱温，可增加柱效（提高灵敏度）

54 提高柱温， 可增加柱效 （提高灵敏度） 目录P611 液相色谱柱: Zorbax 300 StableBond-C3
生物制药 液相色谱柱: Zorbax 300 StableBond-C3 色谱柱尺寸: 4.6 x 150 mm, 5μm 部件号: 肽类/蛋白质: 升温对分离的影响 Peptides/Proteins: Effect of Elevated Temperature 样品: 多肽 流动相: A = 5: 95, 乙腈 : 水, 0.10%三氟乙酸 (V/V%) B = 95: 5, 乙腈 : 水, 0.085%三氟乙酸 (V/V%) 梯度: 在 20 min内 15-53%, 后运行时间 12 min 流速: l.0 mL/min 温度: 室温 -60℃ 检测: UV 215 nm 1. 亮氨酸脑啡呔 2. 血管紧张素II 3. 核糖核酸酶A 4. 牛胰岛素 5. 细胞色素C 6. 溶菌酶 7. 肌红蛋白 8. 碳酸酐酶 1. Leucine Enkephalin 2. Angiotensin II 3. Rnase A 4. Insulin(BOV) 5. Cytochrome C 6. Lysozyme 7. Myoglobin 8. Carbonic anhydrase 提高柱温， 可增加柱效 （提高灵敏度）

55 减小柱径，可增加柱效（提高灵敏度） 目录P611 液相色谱柱: Zorbax StableBond-C8
生物制药 液相色谱柱: Zorbax StableBond-C8 色谱柱尺寸: 150 mm, 5μm 减小毛细管内径来提高 低浓度样品的检测灵敏度 Reducing Capillary ID to Improve Sensitivity for Concentration-Limited Samples 流动相: 60%乙腈/40%水 流速: 内径 0.3 mm-4 uL/min 内径 0.5 mm-10 uL/min 内径 1.0 mm-50 uL/min 内径 4.6 mm-1.0 uL/min 温度: 室温 检测器: UV 254 nm 样品: ng联二苯 (100 nl) 减小柱径，可增加柱效（提高灵敏度）

56 缩短检测器响应时间，可增加柱效（提高灵敏度）
色谱柱技术 – EN 使用小体积色谱柱对于检测器响应时间的重要性 (TE32) 液相色谱柱: ZORBAX StableBond-C18 色谱柱尺寸: 4.6 x 15 mm 3.5µm 部件号: 响应时间: 4.0秒 响应时间: 2.0秒 响应时间: 1.0秒 响应时间: 0.5秒 响应时间: 0.1秒 尿嘧啶 乙酰水杨酸 N-乙酰苯胺 Uracil Acetylsalicylic acid Acetanilide 流动相: 乙腈: 1%甲酸=32: 68 UV: 254 nm 流速: 1.0 mL / min. 30°C 进样量: 5 µL 缩短检测器响应时间，可增加柱效（提高灵敏度）

57 使用高纯硅胶， 可增加柱效 （提高灵敏度） 液相色谱柱 Zorbax Sil Spherisorb Zorbax Rx-Sil
色谱柱技术 – EN 不同硅胶类型比较 (TE1) 液相色谱柱 甲苯 N-苯甲酰苯胺 苯酚 苯甲醇 Zorbax Sil Spherisorb Zorbax Rx-Sil toluene benzanilide phenol benzyl alcohol 流动相: 含0.05%甲醇的二氯甲烷 使用高纯硅胶， 可增加柱效 （提高灵敏度）

58 改变流动相pH， 可增加柱效 （提高灵敏度）
液相色谱柱: ZORBAX StableBond-C18 色谱柱尺寸: 4.6 x 150 mm 5µm 部件号: 有机化学– EN pH对苯胺取代物分析的影响 (ORCH1) 对位甲氧基苯胺 间位苯甲胺 3-氨基-苯甲腈 对位氯苯胺 间位氯苯胺 邻位氯苯胺 p-anisidine m-toluidine 3-amino-benzonitrile p-chloroaniline m-chloroaniline o-chloroaniline pH: 2.5 pH: 7.0 流动相 25mM磷酸: 甲醇=58: 42 流速: 1 mL/min 22°C 改变流动相pH， 可增加柱效 （提高灵敏度）

59 改变流动相pH， 可增加柱效 （提高灵敏度）
液相色谱柱: ZORBAX 300 SB-C8 色谱柱尺寸: 4.6 x 150 mm 5µm 部件号: 生化 –肽– EN 三氟乙酸浓度对于反相肽的分离的影响 (B10) Peptide 流动相: A: 水和三氟乙酸 B: 乙腈和三氟乙酸 0 - 30% B （30 min.内） 流速: 1 mL/min UV: 254 nm 进样量: 6uL 十肽菌素标样S1-S5（疏水性有略微差异） 40°C 0.05%三氟乙酸 0.25%三氟乙酸 改变流动相pH， 可增加柱效 （提高灵敏度） 1.0%三氟乙酸

60 改变有机相%， 可增加柱效 （提高灵敏度） 液相色谱柱: ZORBAX NH2 色谱柱尺寸: 4.6 x 250 mm 5µm
食品分析– EN 流动相强度对单糖和二糖分离的影响 (FD4) 液相色谱柱: ZORBAX NH2 色谱柱尺寸: 4.6 x 250 mm 5µm 部件号: 乙腈/水 70/30 75/25 果糖 葡糖 蔗糖 Palatinose 海藻糖 异麦芽糖 Fructose Glucose Saccharose Palatinose Trehalulose Isomaltose 流动相: 乙腈/水如图 流速: 1 mL/min 示差折光检测器 改变有机相%， 可增加柱效 （提高灵敏度）

61 改变有机相%， 可增加柱效 （提高灵敏度） 液相色谱柱: ZORBAX StableBond-CN
色谱柱尺寸: 4.6 x 150 mm 5µm 部件号: 色谱柱技术 – EN 有机改性剂和pH对于选择性的影响 (TE5) 流动相: 25 mM磷酸缓冲液 流速: 1.0 mL/min. 22°C anisidine m-toluidine 4-cl-aniline 3-aminobenzonitrile 3-cl-aniline 2-cl-aniline 甲氧基苯胺 间位苯甲胺 4-氯-苯胺 3-氨基苯甲腈 3-氯-苯胺 2-氯-苯胺 42%甲醇 ：58%缓冲液 30%甲醇 ：70%缓冲液 改变有机相%， 可增加柱效 （提高灵敏度） pH 2.6 pH 3.0

62 改变键合相， 可增加柱效 （提高灵敏度） 液相色谱柱: ZORBAX Eclipse XDB-Phenyl
ZORBAX Eclipse系列色谱柱的选择性 (TE31) 液相色谱柱: ZORBAX Eclipse XDB-Phenyl 色谱柱尺寸: 4.6 x 150 mm 5µm 部件号: Zorbax Eclipse XDB-苯基 Zorbax Eclipse XDB-C8 Zorbax Eclipse XDB-C18 Uracil Phenol 4-Cl-nitrobenzene Toluene Naphthalene 尿嘧啶 苯酚 4-氯-硝基苯 甲苯 萘 流动相: 甲醇 : 水=70: 30 UV: 254 nm 流速: 1.0 mL / min. 室温 改变键合相， 可增加柱效 （提高灵敏度）

63 改变有机添加剂， 可增加柱效 （提高灵敏度）
目录P613 生物制药 液相色谱柱: Zorbax Extend-C18 色谱柱尺寸: 2.1 x 150 mm, 5um 部件号: 三氟乙酸和氢氧化铵用于肽类 RP-HPLC\ESI-MS分离的选择性比较 Selectivity Comparison of TFA and NH4OH for Peptide RP-HPLC\ESI-MS Analysis 流速: mL/min. 温度: 25℃ 梯度: % B in 20 min. LC/MS: 正离子, ESI- Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12 L/min., 300℃ 样品: 4μL (各50 ng) 三氟乙酸条件: A- 0.1%三氟乙酸水溶液 B-0.085%三氟乙酸 80%的乙腈溶液 改变有机添加剂， 可增加柱效 （提高灵敏度） NH4OH条件: A- 20 mM NH4OH水溶液 B- 20 mM NH4OH 80%的乙腈溶液

64 改变有机添加剂， 可增加柱效 （提高灵敏度）
生化 – 肽 –十肽菌素 – EN 酸对于反相肽的分离的影响(B16) decapeptide 液相色谱柱: ZORBAX 300 SB-C8 色谱柱尺寸: 4.6 x 150 mm 5µm 部件号: 十肽菌素 S1 十肽菌素 S2 十肽菌素 S3 十肽菌素 S4 十肽菌素 S5 decapeptide S1 decapeptide S2 decapeptide S3 decapeptide S4 decapeptide S5 0.1% 三氟乙酸 流动相: A: 含相应的酸的水相 B: 含相应的酸的乙腈 0%-30% B（30 min.内） 进样量: 6µL肽标样 UV: 254 nm 流速: 1 mL/min 40°C 改变有机添加剂， 可增加柱效 （提高灵敏度） 0.025 M H3PO4

65 预混合（重量比混合）: Premixed (w/w)
色谱柱技术 – EN 流动相对色谱的影响(TE40) 液相色谱柱: ZORBAX Eclipse XDB-C8 色谱柱尺寸: 4.6 x 50 mm 3.5µm 部件号: 改变流动相配比方式，可增加柱效 （提高灵敏度） UV: 254 nm 流速: 1 mL/ min. 室温 尿嘧啶 尼帕尔金丁酯 萘 二丙基邻苯二甲酸酯 苊 Uracil Butylparaben Naphthalene Dipropylphthalate Acenaphthene 刻度混合: Dial-a-Mix A: 水 B: 甲醇, 用泵抽取50% B 体积比混合: Vol/Vol 250 mL水 mL甲醇, 用泵抽取100%混合液 定容混合: Mix-to-Mark 250 mL甲醇, 用水定容至500 mL, 用泵抽取100%混合液 预混合（重量比混合）: Premixed (w/w) 200 g甲醇 g水, 用泵抽取100%混合液

66 附录-1 倍数 前缀 符号 1,000,000,000,000,000,000 = exa- E 1,000,000,000,000,000 = peta- P 1,000,000,000,000 = tera- T 1,000,000,000 = giga- G 1,000,000 = mega- M 1,000 = kilo- K 100 = hecto- H 10 = deka- da 0.1 = deci- d 0.01 = centi- c 0.001 = milli- m = micro- u = nano- n = pico- p = femto- f = atto- a ppm ( part per million) = mg/kg, ug/g, ng/mg, pg/ug, mg/L, ug/mL, ng/uL ppb ( part per billion) = ug/kg, ng/g, pg/mg, ug/L, ng/mL, pg/uL ppt ( part per trillion) = ng/kg, pg/g, fg/mg, ng/L, pg/mL, fg/uL mg/mL = 1,000 ppm

67 反相HPLC常用缓冲液 附录-2 缓冲液 pKa pH范围 磷酸盐pK1 2.1 1.1~3.1 柠檬酸盐pK1 3.1 2.1~4.1
磷酸 0.1% 三氟乙酸 0.1% 缓冲液 pKa pH范围 磷酸盐pK ~3.1 柠檬酸盐pK ~4.1 甲酸盐 ~ 4.8 柠檬酸盐pK ~5.7 乙酸盐 ~5.8 柠檬酸盐pK ~6.4 磷酸盐pK ~8.2 羟甲基甲胺 ~9.3 氨 ~10.2 硼酸盐 ~10.2 甘氨酸 ~10.8 1-甲基-哌啶 ~11.3 四氢化吡咯 ~11.5 二乙胺 ~11.5 三乙胺 ~11.7 磷酸盐pK ~13.3 One of the most common problems (mistakes!) made in method development is the use of 缓冲液s at the incorrect pH range. Often methods are written where a 缓冲液 is used outside of its pH range! Be sure to use the proper 缓冲液 for the proper pH. At pH < 2.5, partially ionized strong acids (0.1% phosphoric acid, TFA) can function as effective pH stabilizers. Formate, acetate, TFA and ammonia are volatile, making them a good choice for LC/MS. Volatile (but weak) 缓冲液s can be made by combining salts. Ammonium formate will 缓冲液 weakly between pH and Ammonium acetate will 缓冲液 weakly between pH and These are usually adequate for LC/MS.