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Principle and Application of GC-MS. 2012-09-17. Content. GC-MS &HPLC-MS Instrument and column Sample preparation Preferences Application in my research. GC-MS &HPLC-MS. Preface Differences . 1.Principle polar VS non-polar 2. Mobile phase liquid VS gas 3. Ionization way

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content
Content
  • GC-MS &HPLC-MS
  • Instrument and column
  • Sample preparation
  • Preferences
  • Application in my research
gc ms hplc ms
GC-MS &HPLC-MS
  • Preface
  • Differences

1.Principle

polar VS non-polar

2. Mobile phase

liquid VS gas

3. Ionization way

EI,CI VS ESI, APCI

4.Application

heat stability

principle of gc ms
Principle of GC-MS
  • 物理分离方法;
  • 组份沸点差异 &分配系数的差异;
  • 质谱检测器----电离源----质谱碎片----谱库检

索----匹配度----完成定性。

manufacturer of gc ms
Manufacturer of GC-MS
  • 美国Agilent
  • 日本岛津 Shimadzu
chromatographic column
Chromatographic column
  • Capillary column and packed column
preferences of capillary column
Preferences of capillary column
  • 内径(inner diameter):0.25mm
  • 高柱效,低负荷量,必须分流进样或无分流进样。
  • 用于复杂多组份样品分析。
  • 柱长(column length ):30m
  • 25—30m 中长柱:分离10—50个组份。     50m 长柱:分离大于50个组份或包含有难分离物质。
  • 膜厚(film sickness):0.25—0.33μm
  • 对于流出达300℃的大多数样品(包括蜡、甘油三酯、甾族化合物等)能够很好的分析。
  • 固定液(stationary liquid)

分析时间加倍,分辨率增加40%

stationary liquid
stationary liquid ---“相似相溶”
  • a. 非极性物质—非极性固定液;低沸点组分
  • b. 极性物质—极性固定液;小极性组分
  • c. 极性与非极性混合物—极性固定液;小极性组分
  • d. 易形成氢键物质—极性或氢键型固定液;

不易形成氢键的组分

  • e. 复杂难分离样品—多种固定液混合;
sample preparation
Sample preparation
  • Without water and salt;
  • Organic solvent with low boiling point;
  • 丙酮、正己烷、氯仿、苯等
  • 毛细管柱样品浓度mg/ml级或更低;
  • 进样量----进样方式而定
  • sample derivatization(样品衍生化)
sample derivatization
sample derivatization
  • 硅烷化
  • 酰化
  • (1)乙酰化(体内药物筛选,大多数的临床药物)

(2)三氟乙酰化/五氟丙酰化/七氟丁酰化(苯丙胺类和麻黄碱类)

  • 烷基化(农药和杀虫剂)
  • 目的:改善待测物质的气相色谱性质如热稳定性、分子质量、质谱行为;
preferences of gc ms sample injection
Preferences of GC-MS ----sample injection
  • 自动进样(auto-injection)
  • 手动进样(hand-injection)
split or not split ratio
split or not?split ratio?
  • 分流(split)----主要组分分析;
  • 脉冲分流(pulse split)----允许更大进样量;
  • 不分流(splitless)---- 痕量组分分析;
  • 脉冲不分流(pulse splitless)----允许更大进样量。
carrier gas
carrier gas
  • hydrogen ,nitrogen, argon, helium, Carbon dioxide
  • flow rate:i.d<0.25mm:0.5-3ml/min;
  • 0.32mm: 5-10ml/min;
  • 0.53mm: 5-20ml/min
  • i.d<0.32mm,衡流模式,1.0ml/min;
temperature of inlet
temperature of inlet
  • 考虑样品的稳定性?
  • 样品是否能够瞬间气化?
  • 一般在200~250℃
column temperature program
column temperature program
  • 影响样品分离度的最主要因素;
  • 升温慢可改善分离效果,增加分析的时间;
  • 升温快会加快载气流速,相近组分难完全分离;
  • 柱温极限;
selection principle
Selection principle
  • 在使最难分离的组分尽可能好的分离的情况下,尽可能采取

较低的柱温,但以保留时间适宜,峰形不拖尾为度。

  • 高沸点300-400℃,低于其沸点100-200℃;
  • 沸点200-300℃,中等柱温;
  • 沸点100-200 ℃ ,平均沸点的2/3左右
  • 气体,气态烃等,沸点或沸点以上
ionization source for gc ms
Ionization source for GC-MS
  • 样品分子 ---- 带电的离子 ----离子束----质量分析器
  • EI:NIST标准谱库,70eV,230℃,易挥发 成分
  • CI:软电离技术,EI分析不了的物质,非标准质谱图
  • FAB, ESI, APCI for HPLC-MS
data acquisition
Data acquisition
  • 全扫描(Scan)VS选择离子扫描(SIM)
  • 灵敏度:SIM比Scan的高三个数量级
  • 阈值的设置:为限制采集的峰数
qualitative analysis
Qualitative analysis
  • Library,
  • 一般为probability-based matching(PBM)检索
  • 匹配度≥80,
  • else AMDIS
  • Research
library
library
  • NIST:美国国家科学技术研究所出版
  • NIST/EPA/NIH:美国国家科学技术研究所、美国环保局、

美国国立卫生研究院共同出版

  • Wiley库:3个版本,同一化合物可能有重复的不同来源的

质谱图

  • 农药库、药物库、挥发油库
  • 前三个通用,后三个专用,NIST/EPA/NIH库应用最广泛。
some advice
Some advice
  • 1 EI,70eV for NIST
  • 2 纯化合物的质谱图

Subtract Background(BSB) & Savitzky-Golay

  • 3 匹配度(相似度)& 其他有关信息
influencing factor for library searching
Influencing factor for library searching
  • 仪器调谐方式不对
  • 数据采集时最强峰超量程
  • 采集数据或处理谱图时设定的低质量范围过高
  • 色谱分离不好,或本底峰干扰严重
  • 检索参数的选择(如质量范围等)
quantitative analysis
Quantitative analysis
  • 面积归一化法(area normalization method)
  • 外标法(external standard method)
  • 内标法(internal standard method)
area normalization method
Area normalization method
  • 方法:各组分浓度以面积百分率表示,可确认大概的浓度,但有误差;
  • 要求:
  • 1)所有物质都出峰,各物质响应因子接近,主要物质含量足够大,各峰型都较尖锐且对称;
  • 2)峰高,只要不超过检测的线性范围最大值的一半就行;
external standard method
External standard method
  • 目前应用最广泛的方法之一;
  • 误差来源:主要是进样误差;
  • 解决办法:分析前一定要做面积重复性(即进样重复性)实验
internal standard method
Internal standard method
  • 在样品中添加等体积等浓度的内标物,通过组分与内标峰

的面积比,对组分进行定量;

  • 内标物选择原则:
  • 内标物的峰与试样中的所有成分的峰完全分离;
  • 内标物的峰与目标成分的峰保留时间不应差太远;
  • 内标物具有与分析目标成分类似的化学性质
integrator of chromatographic peak
Integrator of chromatographic peak
  • RTE Integrator
  • ChemStation Integrator
slide32
RTE
  • 相对简单的色谱图(其中的峰很窄且已完全解析)
  • 尤其适用于 target compound analyses(目标化合物分析)
  • 缺省参数
  • 整个色谱图范围内;毛细管色谱柱;总离子流 (TIC)
chemstation integrator
ChemStation Integrator
  • 各种类型色谱图的积分方便,直观,准确
  • 带有难于处理的基线和负峰的色谱图
  • 但自动编辑定量时,用RTE较好
application in my research
Application in my research
  • 6890/5973, Agilent
  • auto sampler (7683B, Agilent)
  • Agilent HP-5MS capillary column (30m×0.25mm i.d., 0.25μm film),
  • 1μl extracts solution (1g/ml, extracts/methanol) & n-Tridecane
  • split ratio 10:1
  • helium (1.0ml/min).
  • column temperature programwas :
  • 60℃(1 min), 60-140℃at 4℃/min, 140℃(10min), 140-155℃at 3℃/min,155℃(2min),155-250℃at 20℃/min,250℃(1min).
  • EI,70eV;
  • Inlet--250℃, ionization source --230℃, MS detector --150℃
  • scan,m/z 40-500.
slide35
参考文献
  • 1. N.Y. Qin, F.Q. Yang, Y.T. Wang, S.P. Li. Quantitative determination of eight components in rhizome (Jianghuang) and tuberous root (Yujin) of Curcuma longausing pressurized liquid extraction and gas chromatography–mass spectrometry[J]. Journal of Pharmaceutical and Biomedical Analysis, 2007,43:486–492.
  • 2.Li, M (Li, Ming), Zhou, X (Zhou, Xin), Zhao, Y (Zhao, Yang), Wang, DP (Wang, Dao-Ping) ,Hu, XN (Hu, Xiao-Na). Quality Assessment of Curcuma longa L. by Gas Chromatogrotphy-Mass Spectrometry Fingerprint, Principle Components Analysis and Hierarchical Clustering Analysis.Bulletin of The Korean Chemical Society[J].2009,30(10):2287-2293.
  • 3. http://www.instrument.com.cn/netshow/SH100320/
  • 4.http://www.home.agilent.com/agilent/home.jspx?cc=CN&lc=chi
  • 5.汪正范,杨树民,吴侔天等. 色谱连用技术[M].化学工业出版社.2001.
  • 6.王光辉, 熊少祥. 有机质谱解析[M]. 化学工业出版社 . 2005.
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