High-performance liquid chromatography (HPLC) is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry to identify, quantify, or purify the individual components of the mixture. HPLC is considered an instrumental technique of analytical chemistry (as opposed to a gravimetric technique). HPLC has many uses including medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products).
The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the number of sample components emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis.
1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in “inject position”, (6′) Switching valve in “load position”, (7) Sample injection loop, (8) Pre-column (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.
The basic concept within the pump head is that the plungers are driven in and out by a motor. At the inlet & outlet ends of the pump, the head is a set of check valves. These valves only allow liquid to flow in one direction, from the mobile phase reservoir to the autosampler, column, and detector. One critical area within the set-up is the plunger seals, they play a crucial role in the overall system performance.
Four basic requirements of a plunger seal designer:
- Leak-free sealing – prevent any mobile phase from leaking from around the sapphire plunger
A leak can cause fluctuations in retention time. Incorrect flow rates may cause system shut-down if enough solvent is present, and if you are using a high pressure mixing gradient system the mobile phase ratios may be incorrect.
- Flexibility- allow for the movement of the sapphire plunger across its surface.
- Chemically resistant – to a wide range of mobile phase solvents and modifiers.
- Strength – robust and durable enough that the frequency of replacement is minimised.