manufacturer :       Pickering

model :                   PCX 5200

annotation :           Dokumente engl .

Why Post- coIumn Derivatization ?

Post-column derivatization , also known as post-column reaction, renders visible certain compounds that are normally invisible. This trick is accomplished after the separation by performing a chemical reaction on the substances that gives them an easily detectable physical property. Typically you use a reaction that produces a strong color or makes a fluorescent product. You can increase the sensitivity of detection by several orders of magnitude in favorable cases. Most reagents are selective for a particular class of substances, so analytes of that class are more easily seen against a complex background. So, post-column derivatization is used to increase sensitivity and selectivity in HPLC analysis.

How Does It Work?

The post-column reaction system mixes the stream of effluent flowing from the HPLC column with a stream of reagent solution. The mixture usually flows through a reactor to allow enough time for the chemical reactions to complete. If the reaction is slow, the reactor may be heated to speed things up. Some reactions need two or more reagents added in sequence. Finally, the mixed streams pass into the detector, typically UV/VIS absorbance or fluorescence. Of course, a practical system requires metering pumps, pulse damper, thermostats, and safety systems to give reliable results.

What Are the Requirements?

• Stability of Reagent. The minimum reagent stability sufficient for routine work is one day. This means that the yield and signal-to-noise ratio for a given sample must remain constant for at least 8 hours.

• Speed of Reaction. The analytical separation is complete when the reagent is mixed with the column effluent. Therefore, in order to minimize band spreading, it is important to keep the time (therefore volume) small between the mixing tee and the detector. If the reaction is slow (in excess of one minute), an elevated temperature can be used to decrease the reaction time.

• Reproducibility. Because the reaction is occurring “on the fly,” as the combined column and reagent stream flows toward the detector, the reproducibility is linked to the flowrate precision of the pumps and to the temperature. Accordingly, even an incomplete reaction will be as repeatable as the retention time for any given species. The completeness of the reaction, then, is not strictly necessary for reproducibility, but it is important for maximum sensitivity.

• Minimal Detector Response of Reagents. The color or background fluorescence of the reagent (or its by-products) represents a continuous noise source. Because the reagent is present in excess relative to the analyte , the analyte’s signal could be obliterated by the reagent’s strong background signal. The baseline noise is proportional to the background signal.

• Solubility. All species must remain in solution, including the combined components of the eluants and the reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors, and foul detector flowcells .

• Uniformity of flow. The baseline noise is a function of the flow-noise in the eluant and reagent pumps. Non-uniform flow causes non-uniform mixing leading to modulation of the background signal which appears as noise. Refractive index noise can be even more objectionable than absorbance noise.

Pickering PCX5200

The PCX5200 does three main things (Figure 1-1):

1. Thermostats the analytical column

2. Delivers the reagent

3. Heats the reaction.

It also has various features to make the analysis more convenient or reliable, and features to protect the instrument itself from accidental damage.

Reagent Delivery

The pressurized reservoir serves two purposes:

1. It protects air-sensitive reagents from oxidation.

2. It helps the metering pump fill consistently by preventing cavitation .

The pump is a constant speed, variable-stroke piston pump. The cam cycle is two seconds, which makes for easy pulse damping.

The reagent pressure gauge does two things:

1. It shows the pump pressure for diagnostic purposes.

2. It absorbs pulses by the spring action of the Bourdon tube. There is a packed-bed restrictor just downstream to provide a load for the pressure gauge so that it will effectively absorb pulsations.

Referring to Fig. 1-1, the anti-siphon valve prevents the gas pressure in the reservoir from forcing liquid through the system with the power off. The post-column pressure gauge is primarily there to monitor the condition of the reactor. There is a 500 psi relief valve in case there is a blockage in the reactor or detector. The standard reactor is a PTFE capillary tube 0.011˝ I.D. wrapped on a heated mandrel. The narrow diameter reduces band-spreading, and the PTFE is corrosion resistant. There is a 100 psi (5 bar ) back-pressure regulator on the exit line from the detector; it suppresses boiling inside the hot reactor and prevents bubbles from forming in the detector flowcell .

Column Protection

Usually, the post-column reagent will immediately damage the analytical column. To prevent this, there is a pressure switch connected between the HPLC pump and the injector that must sense 500 psi (35 bar ) before allowing the reagent pump and reactor to receive power. If at any time pressure is lost because of fault or programmed shutdown, the PCX5200 will gracefully shut itself off until a human turns it back on.

Specifications

Wetted Materials

PCX5200

316 Stainless steel

17-4 PH Stainless steel

FEP

Saran PVDC

Hastelloy -C

Kel -F CTFE

PEEK

Viton

Borosilicate glass

Sapphire

UHMW polyethylene

PCX5200 PEEK

316 Stainless steel

17-4 PH Stainless steel

Viton

FEP

Saran PVDC

Kel -F CTFE

PEEK

Tefzel ETFE

Borosilicate glass

Sapphire

UHMW polyethylene

Ratings

Dimensions:                37 cm x 38 cm x 38 cm (h x w x d)

Weight:                        13 to 15 kg, depending on configuration

Electrical Power:        100–120 V; 50/60 Hz; 1.7 A; 200 W; grounded supply; or

200–240 V; 50/60 Hz; 0.8 A; 200 W; grounded supply

Installation (overvoltage) category II, Pollution degree 2

Environmental:            Indoor use only

Altitude up to 2,000 m (6500 ft)

Ambient temperature 5–40°C (40-104˚F)

Relative humidity 80% @ 31°C, derated to 50% R.H. @ 40°C