飞行时间质谱(TOF-MS)操作规程

飞行时间质谱操作规程
一，靶板清洗程序
1.用丙酮冲洗靶板直到去除所有可见的样品残余
2.用1%甲酸溶液超声10分钟
3.用去离子水超声10分钟
4.用分析纯丙酮超声10分钟
5.用分析纯甲醇超声10分钟
6.放入盒中，自然干燥
二，样品制备
基质选择及配置标准溶液
本实验室现有基质如下：
3-Indoleacetic acid （IAA），3-Hydroxypicolinic acid （HPA），Trihydroxyanthracene （DI），2,5-Dihydroxybenzoic acid （DHB），2',4',6'-Trihydroxyacetophenone monohydrate （THAP），2',6'-Dihydroxyacetophenone （DHAP），2-(4-Hydroxyphenylazo)benzoic acid (HABA)，
根据测试人样品特点，参考相应文献报道，选择合适基质（以下表格仅供参考）
Dissolve 10 mg in 1 mL of mixed solution of acetonitrile and 0.1% TFA (2:3). Most samples such as protein, DNA, sugar, lipid and synthetic high polymer
For 1 mg of high polymer, dissolve 10 mg and 1 mg of NaCl, LiCl or KCl in demineralized water. Polar synthetic high polymer (All become Na, Li or K added ions.)
Gentisic acid, Aldrich 14,935-7 2,5-Dihydroxybenzoic acid (DHB) (C7H6O4:154.1)
Dissolve 108 mg and 16 mg of ammonium citrate dibasic in a 50% acetonitrile solution. Single stranded DNA, RNA of 50 mer or more
For 1 mg of high polymer, dissolve 10 mg in chloroform or THF. Non-polar synthetic high polymer
Dithranol, Sigma D-2773
1,8-Dihydroxy-9[10H]-anthraceno ne
(C14H10O3:226.2) For 1 mg of high polymer,
dissolve 10 mg and 1 mg of
silver trifluoroacetate in
chloroform or THF. Non-polar synthetic high polymer (All become Ag added ions.)
Remarks:
1, 由于本仪器主要用于合成化合物的表征，故以以上两种基质使用最为广泛。

2, 基质溶液应当在每次测试前配制，如需放置隔夜，请储存于闭光、低温条件（冰箱）
三，点样
It is necessary to use methods of dropping to a sample plate that are suitable for different samples or matrices individually. This section describes the following three basic methods.
Dropping method (1): Usually used.
Dropping method (2): Used to drop a large amount of the same sample to samples. (Standard
sample in the auto analysis mode, etc.)
Dropping method (3) : Used for solid samples.
Dropping method (1)
1.Prepare an unused clean sample plate.
2.Drop 0.5μL of sample solution to the specified sample well using a pipette, etc.
Take care not allow the pipette end to touch the plate.
3.Before the crystallization of the sample (within approximately 10 seconds), drop a matrix
solution onto 0.5μL of sample solution.
4.Air dry.
Caution: Exchange the pipette tip every drop irrespective of matrices or sample solutions.
The dropping order of a matrix and that of a sample may be reversed mutually. Dropping method (2)
1.Prepare an unused clean sample plate.
2.Drop 20μL of matrix solution and 1.0μL of sample solution into the sample tube to mix
them.
3.Drop 1.0μL of the mixed solution to the specified sample well using a pipette, etc.
Caution: Prepare a sample solution approximately 20 times stronger than that in the dropping method (1).
Dropping method (3)
1.Prepare an unused clean sample plate.
2.Move a matrix (solid) put in a sample bottle and a sample (solid).
3.Drop an organic solvent to dissolve the matrix and the sample.
4.Drop 2.0μL of the mixed solution to the specified sample well using a pipette, etc.
四，样品分析
测定前的准备工作 打开需要的窗口
3
2
4
Acquisition window
Camera Viewer window
1
6
5
Kompact window
Spectrum Contents window
出现如上几个窗口，可以根据自己的习惯和喜好调整各窗口的位置和大小
Kompact window : 显示数据采集情况 Acquisition window : 设定仪器操作模式、设定和执行数据采集。

Spectrum Contents window :
选择要显示的采集数据 Camera Viewer window : 观察样品基激光照射位点. 设定测定参数 3
2
1
1. 选择测定模式（反射、线性、正离子、负离子）
2. 输入要测定的质量范围.
3. 激光照射频率10或5
4. 点击 Firing 标签. 设定 P .Ext: 值（与样品分子量接近）
放入样品
2
Insert the plate as far as it will
go.
放入样品板
靶板缺角处置于右前方，其两
侧与靶板槽吻合，平行推入。

靶板槽
关闭样品门
待真空度达到 2.0E-3Pa （正离子模式）或 8.0E-4Pa （负离子模式）时可以开始采集数据.
质量校正
1
1.
Display the measurement data (spectrum).
Set to ON only Process on Display Contents window. Click on Apply.
2. Click on Processing through the Kompact window.
3
2
3. Click on Calibration.
4
5
6
7
8
4. Load the reference file.
Click on List references... to display the list.
The reference file for typical standard sample is shown in the table below. Select a reference file according to the molecular weight of sample. Click on Open after selecting a file.
References Nomenclature [M+H]
+
Cyano-Angio2.pos_ref CHCA2*–Angiotensin Ⅱ
379.09, 1046.54 Cyano-Acth.pos_ref CHCA2*–ACTH fragment 18-39 379.09, 2465.1989 Cyano-Insb.pos_ref CHCA2*–Insulin b 379.09, 3495.66 Cyano-Ins.pos_ref CHCA2*–Insulin 379.09, 5730.61 Cytc-Cytc2H.pos_ref Cytochrome C–1/2Cytochrome C*12361.1506, 6181.08 Cytc-Myo.pos_ref Cytochrome C–myoglobin 12361.1506, 16952.5513 Myo-Myo2.pos_ref Myoglobin–Myoglobin2* 16952.5513, 33904.0946 Myo-Ald.pos_ref Myoglobin–Aldrase 16952.5513, 39212.88, Ald-Bsa.pos_ref Albumin–Aldrase 66431.08, 39212.88 Bsa-Bsa2.pos_ref Albumin–Albumin2* 66431.08, 132861.15
Angio2.pos_ref Angiotensin Ⅱ 1046.54 P14R.pos_ref P14R 1533.86
If the peak MS value of measurement standard sample is too deviated from the estimated value, perform the calibration using the mouse specification. 5
9 7 6 4
2 1
1. Display the measurement data and load the reference file.
(Refer to the procedures 1 to 4 of “9.1 Using reference for calibration) 2. Double-click the line of standard sample from the mass table.
3. Match the cursor to the measurement peak. (It is not necessary to match it correctly.)
Measureme nt peak
Cursor
Place the pointer in the Spectrum screen, while pressing and holding the ADJUST button of the mouse, move the cursor to fit it to the peak top. Release the
cursor.
4.Return to the Calibration window and click on (load the cursor).
5.Click on on the Spectrum window (refer to Advanced Guide.).
Perform the same procedures to the peak which other MS value is deviated. （Procedures 2 to 5.）
6.Set the tolerance of calibration (5 in 9.1 Using reference for calibration)
7.Click on Calibrate.
Refer to “9.1 Using reference for calibration” if not succeeded.
8.Return to the procedure “5. of Using reference for calibration”, and perform the calibration
more precisely.
9.Enter the file name and click on Save.
Set the tolerance of calibration.
Set this so that the peak to be calibrated is located within the specified range of in Tolerance. The largest (strongest) peak which was set in this range will be selected in Calibrate of the procedure 6. If there is no peak in the set range, an error may occur. If you set the tolerance too large, an error may occur and the calibration may be largely deviated because other peak is recognized. If it is suspected that the peak of standard sample is deviated from 2 Da or more, proceed to 9.2.
Da : dalton
mDa : 1/1000 milli- dalton
ppt : parts per thousand
ppm : parts per million
Click on Calibrate.
Repeat the following operations if not succeeded.
Click on Undo.
Change the setting in procedure 5.
Click on Calibrate.
Enter the file name.
Click on Save.
After this, select the file from List… if the measurement is performed using the calibration of this measurement mode. Select named calibration in Load:, and click on Load.
样品分析
选择样品
Laser power: 从50起，根据出现的谱图状态进行调整Profile: 1000
Shots: 5
点击.开始采集数据
调整laser power 的依据：
×○△×
(A) (B) (C) (D)
(A): 低⇒提高1 to 3.
(B): 合适⇒可调整能量以得到最佳分辨率
(C): 稍高⇒降低1 to 3.
(D): 太高⇒降低3 to 10.
数据保存
在谱图符合要求后，按Abort键停止测试，按Store键保存谱图。

并参考“放入
样品板”程序，取出靶板。

备注：保持仪器处于OPERATION状态。