Analytical Method Development - 分析方法开发

hunter

GC Method Development
http://www.agilent.com/cs/librar ... d%20Development.pdf

What to Consider
The Sample
Method of injection
Inlet
Detector
Detector
Carrier Gas
Column
COMPOUND REQUIREMENTS FOR GC
Only 10-20% of all compounds are suitable for GC
analysis
The compounds must have:


Sufficient volatility
Group/Presentation Title
Agilent Restricted
Month ##, 200X
Page 3


Sufficient volatility


Thermal stability
NO
Inorganic Acids and Bases
Be mindful of salts!
Sample Considerations
1.   Sample matrix
residues?
dirty samples?
2.   Analyte Composition
1.
Isomers?
1.
Isomers?
2.     Polar vs. non-Polar?
3.     Organinc Acids?
4.     Light Gases?
5.     Nobel Gases?
6.     Halogens?
Sample Residues
Semi-volatile residues
Bake out
Back flush
Non-volatile residues
Guard column
Guard column
Bake out
Back flush
Dirty Samples
Sample clean up?
Back flush

http://www.chem.ucla.edu/~bacher/General/30BL/gc/theory.html

Gas Chromatography (GC or GLC) is a commonly used analytic technique in many research and industrial laboratories for quality control as well as identification and quantitation of compounds in a mixture. GC is also a frequently used technique in many environmental and forensic laboratories because it allows for the detection of very small quantities. A broad variety of samples can be analyzed as long as the compounds are sufficiently thermally stable and volatile.
                How does gas chromatography work?
                Like for all other chromatographic techniques, a mobile and a stationary phase are required for this technique. The mobile phase (=carrier gas) is comprised of an inert gas i.e., helium, argon, or nitrogen. The stationary phase consists of a packed column where the packing or solid support itself acts as stationary phase, or is coated with the liquid stationary phase (=high boiling polymer). Most analytical gas chromatographs use capillary columns, where the stationary phase coats the walls of a small-diameter tube directly (i.e., 0.25 m film in a 0.32 mm tube).
                The separation of compounds is based on the different strengths of interaction of the compounds with the stationary phase (“like-dissolves-like”-rule). The stronger the interaction is, the longer the compound interacts with the stationary phase, and the more time it takes to migrate through the column (=longer retention time). In the example above, compound X interacts stronger with the stationary phase, and therefore lacks behind compound O in its movement through the column. As a result, compound O has a much shorter retention time than compound X.
                Which factors influence the separation of the components?
                       
                        1. Boiling point
                       
                        The boiling point of a compound is often related to its polarity (see also polarity chapter). The lower the boiling point is, the shorter retention time usually is because the compound will spent more time in the gas phase. That is one of the main reasons why low boiling solvents (i.e., diethyl ether, dichloromethane) are used as solvents to dissolve the sample. The temperature of the column does not have to be above the boiling point because every compound has a non-zero vapor pressure at any given temperature, even solids. That is the reason why we can smell compounds like camphor (0.065 mmHg/25 oC), isoborneol (0.0035 mmHg/25 oC), naphthalene (0.084 mmHg/25 oC), etc. However, their vapor pressures are fairly low compared to liquids  (i.e., water (24 mmHg/25 oC), ethyl acetate (95 mmHg/25 oC), diethyl ether (520 mmHg/25 oC)).
                       
                        2. The polarity of components versus the polarity of stationary phase on column
                       
                        If the polarity of the stationary phase and compound are similar, the retention time increases because the compound interacts stronger with the stationary phase. As a result, polar compounds have long retention times on polar stationary phases and shorter retention times on non-polar columns using the same temperature. Chiral stationary phases that are based on amino acid derivatives, cyclodextrins and chiral silanes are capable of separating enantiomers because one enantiomer interacts slightly stronger than the other one with the stationary phase, often due to steric effects or other very specific interactions. For instance, a cyclodextrin column is used in the determination of the enantiomeric excess in the chiral epoxidation experiment (Chem 30CL).
                       
                        3. Column temperature
                       
                        A excessively high column temperature results in very short retention time but also in a very poor separation because all components mainly stay in the gas phase. However, in order for the separation to occur the components need to be able to interact with the stationary phase. If the compound does not interact with the stationary phase, the retention time will decrease. At the same time, the quality of the separation deteriorates, because the differences in retention times are not as pronounced anymore. The best separations are usually observed for temperature gradients, because the differences in polarity and in boiling points are used here (for examples see the end of the chapter)
                       
                        4. Carrier gas flow rate
                       
                        A high flow rate reduces retention times, but a poor separation would be observed as well. Like above, the components have very little time to interact with the stationary phase and are just being pushed through the column.
                       
                        5. Column length
                       
                        A longer column generally improves the separation. The trade-off is that the retention time increases proportionally to the column length and a significant peak broadening will be observed as well because of increased longitudinal diffusion inside the column. One has to keep in mind that the gas molecules are not only traveling in one direction but also sideways and backwards. This broadening is inversely proportional to the flow rate. Broadening is also observed because of the finite rate of mass transfer between the phases and because the molecules are taking different paths through the column.
                       
                        6. Amount of material injected
                       
                        Ideally, the peaks in the chromatogram display a symmetric shape (Gaussian curve). If too much of the sample is injected, the peaks show a significant tailing, which causes a poorer separation. Most detectors are relatively sensitive and do not need a lot of material in order to produce a detectable signal. Strictly speaking, under standard conditions only 1-2 % of the compound injected into the injection port passes through the column because most GC instruments are operated in split-mode to prevent overloading of the column and the detector. The splitless mode will only be used if the sample is extremely low in concentration in terms of the analyte.
                       
                        7. Conclusion
                               
                                High temperatures and high flow rates decrease the retention time, but also deteriorate the quality of the separation.
                               
                               
                                        Which detectors are used?
                               
                                1. Mass Spectrometer (GC/MS)
                       
                        Many GC instruments are coupled with a mass spectrometer, which is a very good combination. The GC separates the compounds from each other, while the mass spectrometer helps to identify them based on their fragmentation pattern (see Mass Spectrometry chapter).
                       
                        2. Flame Ionization Detector (FID)
                       
                        This detector is very sensitive towards organic molecules (10-12 g/s = 1 pg/s, linear range: 106-107), but relative insensitive for a few small molecules i.e., N2, NOx, H2S, CO, CO2, H2O. If proper amounts of hydrogen/air are mixed, the combustion does not afford any or very few ions resulting in a low background signal. If other carbon containing components, are introduced to this stream, cations will be produced in the effluent stream. The more carbon atoms are in the molecule, the more fragments are formed and the more sensitive the detector is for this compound. Unfortunately, there is no direct relationship between the number of carbon atoms and the size of the signal. As a result, the individual response factors for each compound have to be experimentally determined for each instrument. Due to the fact that the sample is burnt (pyrolysis), this technique is not suitable for preparative GC. In addition, several gases are usually required to operate a FID: hydrogen, oxygen (or compressed air), and a carrier gas.
                       
                        3. Thermal Conductivity Detector (TCD)
                       
                        This detector is less sensitive than the FID (10-5-10-6 g/s, linear range: 103-104), but is well suited for preparative applications, because the sample is not destroyed. The detection is based on the comparison of two gas streams, one containing only the carrier gas, the other one containing the carrier gas and the compound. Naturally, a carrier gas with a high thermal conductivity i.e., helium or hydrogen is used in order to maximize the temperature difference (and therefore the difference in resistance) between two filaments (= thin tungsten wires). The large surface-to-mass ratio permits a fast equilibration to a steady state. The temperature difference between the reference and the sample cell filaments is monitored by a Wheatstone bridge circuit (the student learnt about this circuitry in physics!).
                       
                        4. Electron Capture Detector (ECD)
                       
                        This detector consists of a cavity that contains two electrodes and a radiation source that emits  -radiation (i.e., 63Ni, 3H). The collision between electrons and the carrier gas (methane plus an inert gas) produces a plasma-containing electrons and positive ions. If a compound is present that contains electronegative atoms, those electrons will be “captured” to form negative ions and the rate of electron collection will decrease. The detector is extremely selective for compounds with atoms of high electron affinity (10-14 g/s), but has a relatively small linear range (~102-103). This detector is frequently used in the analysis of chlorinated compounds i.e., pesticides (herbicides, insecticides), polychlorinated biphenyls, etc. for which it exhibits a very high sensitivity.
                       

hunter

http://www.maplevoice.com/bbs/forum.php?mod=viewthread&tid=472&extra=page%3D1&mobile=2

1.        First, you shoud know analytes structure, solubility and choose the detector based on analytes (or your interest) if the analytes are uv active use UV or RID, LSD and MSD etc.,HPLC method development start with literature survey to get rough idea about development. start with solubility of the analytes,if it is soluble in polar solvents we can try with reverse phase if is is not we should use normal phase chromatography. Mostly many organic drug molecules are polar or mid polar so we usually go for RPC  2.        selection of wavelength by PDA (not always wavelength max, based on related compounds, UV cut off of mobile phase, to avoid matrix intereferencese etc.) . if the analytes are not UV active then there is dervatisation method.   3.        column selection (start with C18 inertsil and short column (100mm or 150 mm) if it is revesed phase or u can use GL Sciences – Inertsil ODS, waters Xbridge, Agilent Zorbax eclips columns or phenomenex Gemini etc)  4.        Mobile Phase: Gradient setting(Always develope the method by using gradient than isocratic). use buffer or modifier if the analytes are ionisable because ionisable compounds will show as two peaks or split peak as it is existing in two form (HA and A-); so if you buffered the mobile phase into basic or acidic the analyte will go to single form. maximum use acidic pH (2-4) as the mostly column will hydrolyse more in basic condition  HA------------>H+ + A-  0.1% TFA, H3PO3, H2SO4, 1-2 % Acetic acid are the simple modifiers to restrict the pH into acidic side to improve the peak shape) and well known phasphate buffer (10 mmol – 100 mmol phasphate buffer), optimum is 30-50 mmol start with mobile phase composition Use water & Methanol or ACN according to solubility in 50:50 ratio. feed other parameter like wavelength, ambient Temp, 1ml/min Flow rate. About sample preparation for assay preparation use conc in between 100-500 ppm as per peak response. keep the peak response below 1000mV = 1V or 1000 mAU= 1AU to keep the UV detector linearity. If there are more than 10 molecule in particular product / sample go for gardient method for better resolution & short run time.you can start with simple greadient  Time(min)---------%A(Water or Buffer)-------------%B (Organic Modifier-ACN,MeOH) 0---------------------80---------------------------------------20 25-------------------20---------------------------------------80 35-------------------20---------------------------------------80 40-------------------80---------------------------------------20 50-------------------80---------------------------------------20  while developing any method always follow ICH parameter abt Tailing factor, Capacity factor, Assymetry, thereotical plates.Change MP composition ,Column. Once you get better result then for sharpness or to save time adjust flowrate & increase Temp.  If our desired peak is not retain on water /ACN/Methanol composition MP then go for Buffer. Phosphate buffer is widly use buffer for its PKa value,easily Available, gives more Hydrophillic effect, stable or non degradable for 2-3 days.  ...............Before starting any method development on   5.        HPLC Flush the system with water, check pressure, saturate column with MP, check callibration status, lamp Hours.  Always keep data about whatever trials you have done during method development.  Method Development is very challenging task so always be logical before any changes in any parameter.  Mostly For HPLC method Development 10 gm sample is enough but for overall development max 100 gm sample is sufficient.  Its a tentative quantity.  After method development method validation must be done. use ich Q2(R2) for method validation  http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html

hunter

http://www.maplevoice.com/bbs/forum.php?mod=viewthread&tid=472&extra=page%3D1&mobile=2
Instructions to develop a HPLC method for Unknown Samples:

1
Search the available literature to see if there already exists a method for the same analyte or one very similar. You should examine chemical journals and reference books of HPLC results for methods for the chemical of interest. The process of developing a new method is time consuming. It is much more efficient to use previously developed method.

2
Perform a solubility study on the analyte or group of analytes. The solubility characteristics are vital in selecting a column material, solvent and particular mode of HPLC to undertake.

3
Select the best stationary phase or column material to best match your analytes. The stationary phase is contained in the column used in HPLC analysis. It is the main component that separates chemical compounds from each other. The column packing material is a silica based material that possesses bond functional groups that interact with the chemical compounds as they pass through the column. Each chemical interacts differently and is attracted to the functional group to varying degrees. By the time the unknown sample travels the length of the column, the chemicals that have very little attraction to the column material will come out first followed by each of the other chemicals that make up the sample in order of degrees of interaction.The degree of attraction for the stationary phase (packing material) will weigh heavily on the particular material selected.

4
Choose a solvent for the analysis. The solvent must provide some solubility to the analyte and work well with the stationary phase. The solvent is an organic or polar liquid that runs through the whole system and transports the sample as it flows. The solvent transports the sample through the system and aids in the separation process. The analyte interacts with the column material as it passes through the column. The degree of interaction with the column material causes a separation of the chemicals from one another. As the solvent carries the sample through the column, separation continues to occur between the chemical compounds in the sample and by the end of the column an individual band in the solvent stream will represent each chemical compound. This process provides the separation of the analytes from each other.

5
Decide on the effective column length to achieve the degree of separation desired. The longer the column is, the greater the separation. Typical column lengths are 50 cm, 100 cm, 150 cm and 250 cm.

6
Consider which internal diameter of the column is most appropriate for your application. The internal diameter of the column defines the maximum sample size that can be handled by the column without overloading the column. The smaller the internal diameter, the less solvent is required to run the analysis and the less sample is required. This can present a large cost savings to a company that intends to run the method often.

7
Select the best detector for your analyte. There are a multitude of detectors available each with its own special characteristics. The detector responds to the chemical compounds present in the solvent stream as it passes from the column on its way to the waste container. Examples of the types of detectors that are common include fluorescence detectors, Ultraviolet/Visible spectrophotometers and refractive index spectrometers. Depending on the information you desire from the analysis, you should select the detector that will provide it.

8
Assemble the instrument with the chosen characteristics and begin a study investigating the best flow rate and sample size to achieve the results you desire. The flow rate selected is the flow rate that gives the quickest results with the best separation of chemical compounds. As the flow rate increases so does the pressure of the system, so you must make trade offs to achieve the best method. Sometimes you must run at a lower flow rate to stay within the limits of the solvent pump and accept the longer analysis time.

Tips & Warnings

Use only Spectro or HPLC solvents. Trace impurities can concentrate on the column.

Choose only a solvent that will not damage the instrument or column material.

Investigate only one variable at a time. Once you have finished that study hold that variable and begin a study changing the next variable.

hunter

hunter 发表于 2016-2-14 23:37
http://www.maplevoice.com/bbs/forum.php?mod=viewthread&tid=472&extra=page%3D1&mobile=2
Instructions ...

BOOK: Organic analytical chemistry : https://books.google.ca/books?id ... e&q&f=false