Circumstances of HPLC Laboratory

[syx]

Dear Members,
How is the best condition for HPLC room (%RH, temperature)?
What is the effect of room’s circumstance to HPLC? Any literature is pleased.

Best regards,
SYX

[Consumer Products Guy]

It's important to heat your column a few degrees over your highest room temperature, to ensure a constant environment (HPLC with temperature control). Venting your solvent reservoirs and solvent waste containers to an exhaust hood (or having the HPLC in an exhaust hood would not be a bad idea.

[Mark Tracy]

I am also a big supporter of column thermostats. You can get heat and cool column thermostats that will maintain at or below room temperature. Of course they cost more than heat-only designs. A moderate temperature of 25 or 30 C is good for most situations. Columns last longer near room temperature.

Your detector is also temperature sensitive. Most modern designs have a very small temperature coefficient, but some older models display a wave in the baseline that tracks room temperature. If you observe this, you can protect your instrument from hot or cold air blowing on it from your air conditioning system.

For relative humidity, there should be a specification in the operator's manual in the section on installation or site requirements. Usually <90% R.H, or non-condensing.

[Uwe Neue]

A good rule of thumb is that retention times (in isocratic RP) will change by 10% for a 5 degrees change in temperature.

[pawan ratra]

HPLC is used for both normal and reverse phase. the organic solvents used are volatile and change in the conc. of mobile phase may take place at higher temperature. This factors should also be kept in mind.

[Uwe Neue]

No, the volatility of the solvent is irrelevant for a change in composition, since you are heating the solvent under high-pressure conditions. Volatility of solvents may only be important for creating gas bubbles in the detector, but this can be fixed as well, if necessary.

[amaryl]

HPLC is used for both normal and reverse phase. the organic solvents used are volatile and change in the conc. of mobile phase may take place at higher temperature. This factors should also be kept in mind.

As long as ur mobile phase is not exposed to environment its composition won't change. More so addition of water or buffer containing water will add hydrogen bonding to solvents and decrease the volatility. keeping ur column at high temperature than room temp will decrease the viscosity that will result in low back pressure of column.

amaryl

[pawan ratra]

Have you ever worked with HPLC instrument kept at temperature condition 35C (e.g. uncontrolled room temperatures in summers in countries like Africa).
I have experienced the change in retention time due to Change in mobile phase composition with time.

Thus conditions of temperature and %RH of HPLC room should be maintained at certain levels.

[amaryl]

Have you ever worked with HPLC instrument kept at temperature condition 35C (e.g. uncontrolled room temperatures in summers in countries like Africa).
I have experienced the change in retention time due to Change in mobile phase composition with time.

Thus conditions of temperature and %RH of HPLC room should be maintained at certain levels.

well i agree with you working with uncontrollable room temperature will affect your separation both K and separation factor. Does it even happens even when your column is thermostated?

regards

amaryl

[syx]

No, the volatility of the solvent is irrelevant for a change in composition, since you are heating the solvent under high-pressure conditions. Volatility of solvents may only be important for creating gas bubbles in the detector, but this can be fixed as well, if necessary.

What about moist absorption by mobile phase components in reservoir, especially for unaqueous system. May it need special cap with dehumidifier (moist absorber) that connected to vent hole of the resevoir? Does anyone know the supplier of this kind of cap?

[unmgvar]

Syx,

For organic systems which are very influenced by their level of water content (even with small %) it is best to never use a dry solution.
The best way is to use "half" saturated solvents. some method were even developed in such a way that you will need to fully saturate the solvent. in any case working with the dry solvent only will be very hard and not very repeatable from injection to injection.
dry solvent are also very susceptible to air, so make sure that they were very well degassed throu sonication.

[Victor]

I think what Pawan Ratra means is the loss of the most volatile component from a mixture of solvents held in a single reservoir. Yes this is distinctly possible and is a separate issue from the effect of temperature on the retention of sample components when the mobile phase has constant composition.

[Uwe Neue]

I agree with unmgvar that the best approach to control retention in normal phase chromatography is to work with Solvents that are half-saturated with water. I have also seen the use of drying tubes, if somebody is forced to work with very dry hexane, for example. However, applications of this nature are rare.

[DR]

Aside from keeping your (for example) ether from preferentially evaporating out of your hexanes, minor fluctuations in room temperature are rarely a problem. Larger fluctuations can be a problem, especially if you're using a RI detector (big problem).

[Mark Tracy]

One way to reduce evaporation from the solvent reservoir is to use a closed system. This can also reduce water absorbtion. Apply a static pressure of about about 2-5 psi (0.1-0.3 bar) of helium; one cylinder should last a month or more if you have no leaks. You can get the reservoir caps, manifolds, etc. from several suppliers of chromatography accessories.