1. What are the essential components of an HPLC system?
HPLC instrument consists:
An eluent delivery system (= pump)
An injector, a column
A detector and a data evaluation system
2. What is the difference between an isocratic and a gradient system?
It can be distinguished easily. If there is only one inlet tube for the eluent, it is an isocratic instrument, and, there are two or more are present, its a gradient system.
3. What is the gradient HPLC system?
In a gradient system, two or more solvents are continuously mixed during the separation.
4. What are the types of gradient mixing?
A. When mixing of solvent happens before the pump by a proportional valve, it is a low pressure gradient.
In a low pressure gradient system, the mixing happens in the normal pressure or low pressure side of the device before the pump.
B. When the mixing happens after the pump on the high pressure side, mixing takes place in a mixing chamber where the solvents of both pumps meet. Such an instrument has a high pressure gradient.
5. What are the types of sample injector?
A. Sample introduction with a hand injector or a manual valve
B. Sample introduction with an autosampler
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6. What is the function of the HPLC column?
The HPLC column is the heart of the system. The function of the HPLC column is to separate the compound with various separation mechanisms.
7. What is the function of the HPLC column oven?
Function of the column oven is to maintain a constant temperature of the column and support to get reproducible results.
8. What is the most commonly used detector in HPLC systems?
UV detector and diode array detector (also called as PDA – Photodiode Array).
9. What are the various types of detectors used in HPLC systems?
(i) UV-Visible HPLC Detector
(ii) PDA Detectors (Diode Array Detector or Photo Array Diode Detector) HPLC detector
(iii) Refractive-Index HPLC Detector
(iv) Evaporative Light Scattering Detector (ELSD) HPLC Detector:
(v) Multi-Angle Light Scattering Detector (MALS) for HPLC:
(vi) HPLC-Mass Spectrometer (HPLC-MS) Detector
(vii) HPLC Conductivity Detector:
(viii) HPLC-Fluorescence Detector:
(ix) Chemiluminescence HPLC Detector:
(x) HPLC Optical Rotation Detector or Chiral Detector
(xi) Electrochemical (Amperometric) Detector for HPLC
(xii) HPLC Photoconductivity Detectors
(xiii) HPLC Infrared (IR) Detectors
(xiv) Laser-Induced Fluorescence Detector
(xv) Radioactivity Detector
(xvi) HPLC-NMR Detector
10. What is used to transfer the mobile phase from one module to another?
The mobile phase is transferred from one module to the another module through capillaries made up of stainless steel or PEEK (polyetheretherketone).
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11. What is the internal diameter of the HPLC capillaries?
The typical internal diameter of the HPLC capillaries between pump and injector is 0.5-1 mm.
The typical internal diameter of the HPLC capillaries after the outlet of the injector is less than 0.2 mm.
12. What is the internal diameter of the HPLC capillaries when back pressure is required to achieve?
Detectors such as fluorescence detectors need some back pressure for adequate operation which can be achieved with a 0.1-0.2 mm capillary internal diameter.
13. What are restrictor capillaries, sometimes also simply called or restrictors?
A restriction capillary or restrictors are the capillaries with very narrow internal diameter, that restrict the mobile phase flow. It has the capability to closely mimic the normal operating conditions by generating back-pressure of 1k to 2k psi.
14. What is the reason for using interconnection pieces of HPLC from the same manufacturer?
Interconnection pieces of HPLC such as ferrules and fittings should be used from the same manufacturer, because while using different make, they may have different dimensions and internal diameters leading to a small dead volume. This can cause abnormality during separation..
15. What is the typical HPLC startup procedure?
A. Flush system without column at a flow rate of 1 ml/min with a 50/ 50 mixture isopropanol/
water for about 10 min.
B. Inject the mobile phase a few times in order to ensure that the old eluent or impurities are removed from the sample injection system.
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16. What precautions should be considered before the first sample run in the HPLC?
i. Once the mobile phase is prepared and run in the system, leave the HPLC system for a little time to equilibrate to flush impurities or dirt if any out of the column.
ii. Dissolve samples adequately as per method of analysis.
iii. No particles should remain in the sample, use membrane filtration as per method.
iv. Ensure that your dissolved sample should not precipitate in the mobile phase.
v. Inject a standard and take a look at the chromatogram.
vi. Ensure that the baseline is stable with no drift and peaks are symmetrical.
vii. Ensure that the chromatogram after the second injection is identical to the first one.
17. How to flush the new reversed-phase column?
While installing a new reversed-phase column, flush it with acetonitrile or methanol before use it for the first run.
18. How to ensure that system is free of buffers?
Flush the system first with an isopropanol/ water mixture and then with methanol.
19. What is the procedure to temporarily stop the HPLC (When you are aware that you will run the HPLC on the next day or after a pause)?
When you know that you will run the HPLC (same setup) after some time, shut down all components of the instrument except the pump. Keep the pump running at a lower flow rate about 0.1-0.3 ml/min.
Ensure that sufficient mobile phase is available. Do not run HPLC dry. While resuming the HPLC again, adjust the flow as per the method.
20. What is the procedure to stop the HPLC?
When you want to stop HPLC for a longer duration, flush the system using water to remove the buffer out from the system. Followed by flushing the HPLC using 20-30 ml methanol or acetonitrile.
Store the column using acetonitrile or methanol. Close the column with end fittings to prevent drying of the stationary phase.