Operation of Waters HPLC
1. Introduction
This HPLC possesses all typical features of HPLC. The solvent delivery system is capable pumping four different solvents, which make a gradient solvent delivery system. The Photo-Diodearray Detector is featured providing a 3-dimentional signal, a continuous changing with time and wavelength.
(An UV detector is providing a fixed or several fixed wavelength.) The HPLC is controlled by the Milenium software on a 486-66 computer.
HPLC can be used for analysis of organic or polymeric samples, so long as the samples are dissolvable in the eluent used for the delivery. The sensitivity is fully depending on the detector and the samples.
The General Consideration of HPLC (a section for Graduate Student Handbook) can be a reference for further reading or general guide.
2. Structure of the HPLC
Solvent delivery system:
i. pump, dual pump with four solvent lines
ii. pump controller, store up to 12 programmable methods with 10 different curved gredient preset.
Diodearray detector:
Wavelength range covers from 190 nm to 650 nm, with resolution of 1.3 nm.
Autosampler.
hold up to 96 sample vials, make variable volume injection without changing the sample loop.
3. Operation of the HPLC
i. Sample preparation
All the samples should be filtrated, make sure that there is no particles in the sample before making injections. The sample solution should be prepared with the solvent same as the eluent used in the elution.
ii. Eluent preparation
Eluent should be filtrated and degassed for all buffers and solvents except those HPLC grade solvent, e.g. the most commonly used methanol and acetonitrile.
iii. Turn on the HPLC
When all solvents are ready, turn all the powers on with squential of pump, detector and autosampler, and finally computer. User has to wait for the bus initialization finished before doing anything further.
iv. Prime the HPLC
Counterclockwise lose the prime valve. Draw the solvent with a syringe and remove all the air bubbles. If possible, push back the solvent to the solvent line, until no more bubbles can be seen when draw the solvent. When finish the prime, close the prime valve. Now you can turn on the pump and test if there is any leaking. If everything works all right bring up the column.
v. Bring up the column
Bring up (or down the column has to be done with a slow transition, at least 15 minutes. At any time, user has to bring up or down the column before method development or analysis. Any big jump from one solvent to another will shorten the lifetime of the column or in an extreme case, damage the HPLC.
See the section below for detail of the method development.
vi. Sample analysis
Switch the method from the method that brings up the column to your analytical method.
Making injections of samples by using HPLC syinge, that bares a blunt tip.
For normal injections, 20-50
ml should be all right. Of course, adjustment should be done based on the sample concentration. An oversized sample will damage the column and unfavor the resolution of the chromatogram.vii. Turn off the HPLC
Turn off the HPLC in a reverse way of turning it on. But user has to first wash the column clean then bring down the column to fully organic solvent before shutting down the pump. Storing the column in buffers for a prolonged time will shorten the lifetime of the column.
4. Method development
Try methanol/water or acetonitrile/water first. If any an isocratic method can do a good job, do not use gradient. Generally, a 65% methanol can be a good starting point.
Use a known method. This could be a most convenient way of analyzing a similar or series compounds by using the conditions in the published paper, thesis, or private communication.
Develop your own method. If none of the above method works for your sample, modify an existing method, by changing a new column, adding new buffers, changing the concentration of the buffers, changing the gradient or flow rate will help. By using the combination of them.
The criterion to judge a good method for the analysis should be small k', good Rs, for example, 1.5 or 2.
5. Computer control
To operate the HPLC user has to open an account
i. login in
ii. change into your own directory
iii. create an instrument method (or open an existing one).
iv. define detector parameter
choose an observation wavelength
sampling rate
AUFS
v. define pump parameters
isocratic or gradient
flow rate
vi. save the instrument method
ix. Accessing Quick Set Control Entering information in the sample loading table
Entering the run mode Choosing a single sample or multiple sample introducing processing method.
x. equilibrate the system
6. Applications
Quantitative analysis needs response curve first by using an internal standard. At certain fixed conditions, however, without using an internal standard can still give satisfied results, if the analyses are carried out within reasonable long time, for example, days, maybe weeks, if all other conditions are remained the same. Please note that the sample should be reasonably clean.
7. Special notes and Trouble shooting
i. At any time buffer solution should not switch to a pure organic eluent quickly.
Eluents, samples must be filtrated before using.
ii. Samples to be injected must be homogeneous.
iii. pH value of samples and eluents must be within the range of the working conditions of the column.
iv. Minimum transition time from one eluent to another should be 15 minutes.
v. Too high pressure of the HPLC indicates a blocked guard column.
vi. A too low pressure of the HPLC hints that solvent delivery system has an opening.