Analyte Loss EPA 525.2

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

75 posts Page 4 of 5
Just to add to the potential confusion: http://pubs.acs.org/cen/safety/20001218.html
Ethyl acetate may contain peracetic acid.
Steve Reimer wrote:
Just to add to the potential confusion: http://pubs.acs.org/cen/safety/20001218.html
Ethyl acetate may contain peracetic acid.



Thanks Steve. Peroxides (in high enough concentration) will degrade PAHs.
Don Shelly
LGC Standards
Is there a possibility of PAH's sticking to the glass of the vials?

We store our DCM extracts (Liq-liq extraction of (drinking-)water)) in a fridge at 5+/-3°C and do not have any problems over short term (2weeks max). If there is a difference it usually is napthalene that is getting low. We use 2ml amber vials that are silanized. The screw-caps have a PTFE seal on the inside to prevent the solvent from having contact with the silicone septum.

What we do find is a difference in recovery when the extracted standards are compared to standards made by spiking the same amount of calibration-standard directly into DCM. The solvent volume equals the volume recovered when using Liq-liq extraction and internal standard is added so even small variations should be corrected for.
Instead of 10ng/l the external standards only give 5-6ng/l. The extracted standards are fine (compared to an extracted calibration).
BMU_VMW wrote:
Is there a possibility of PAH's sticking to the glass of the vials?

We store our DCM extracts (Liq-liq extraction of (drinking-)water)) in a fridge at 5+/-3°C and do not have any problems over short term (2weeks max). If there is a difference it usually is napthalene that is getting low. We use 2ml amber vials that are silanized. The screw-caps have a PTFE seal on the inside to prevent the solvent from having contact with the silicone septum.

What we do find is a difference in recovery when the extracted standards are compared to standards made by spiking the same amount of calibration-standard directly into DCM. The solvent volume equals the volume recovered when using Liq-liq extraction and internal standard is added so even small variations should be corrected for.
Instead of 10ng/l the external standards only give 5-6ng/l. The extracted standards are fine (compared to an extracted calibration).


What procedure do you use to make the calibration standards in DCM? What solvent is the stock standard made in?

Does your extraction involve separatory funnels? What are they made of? What is your spiking technique? What solvent is your spike in?

Yes, PAHs love glass, but their love of solvent is greater.

Don
Don Shelly
LGC Standards
Don Shelly wrote:
BMU_VMW wrote:
Is there a possibility of PAH's sticking to the glass of the vials?

We store our DCM extracts (Liq-liq extraction of (drinking-)water)) in a fridge at 5+/-3°C and do not have any problems over short term (2weeks max). If there is a difference it usually is napthalene that is getting low. We use 2ml amber vials that are silanized. The screw-caps have a PTFE seal on the inside to prevent the solvent from having contact with the silicone septum.

What we do find is a difference in recovery when the extracted standards are compared to standards made by spiking the same amount of calibration-standard directly into DCM. The solvent volume equals the volume recovered when using Liq-liq extraction and internal standard is added so even small variations should be corrected for.
Instead of 10ng/l the external standards only give 5-6ng/l. The extracted standards are fine (compared to an extracted calibration).


What procedure do you use to make the calibration standards in DCM? What solvent is the stock standard made in?

Does your extraction involve separatory funnels? What are they made of? What is your spiking technique? What solvent is your spike in?

Yes, PAHs love glass, but their love of solvent is greater.

Don


I wonder if the quality of the vials will make a difference. I remember when we were trying to do EPA 535 that if we had any scratches what so ever in the Zymark tubes we would lose 50% of some of the analytes just during blowdown. I have another compound we work with that when stored in a Pyrex 10ml volumetric it will last for a year, but when stored in a plain glass 2ml autosampler vial it will go bad within a week.
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
Don Shelly wrote:
BMU_VMW wrote:
Is there a possibility of PAH's sticking to the glass of the vials?

We store our DCM extracts (Liq-liq extraction of (drinking-)water)) in a fridge at 5+/-3°C and do not have any problems over short term (2weeks max). If there is a difference it usually is napthalene that is getting low. We use 2ml amber vials that are silanized. The screw-caps have a PTFE seal on the inside to prevent the solvent from having contact with the silicone septum.

What we do find is a difference in recovery when the extracted standards are compared to standards made by spiking the same amount of calibration-standard directly into DCM. The solvent volume equals the volume recovered when using Liq-liq extraction and internal standard is added so even small variations should be corrected for.
Instead of 10ng/l the external standards only give 5-6ng/l. The extracted standards are fine (compared to an extracted calibration).


What procedure do you use to make the calibration standards in DCM? What solvent is the stock standard made in?

Does your extraction involve separatory funnels? What are they made of? What is your spiking technique? What solvent is your spike in?

Yes, PAHs love glass, but their love of solvent is greater.

Don


I wonder if the quality of the vials will make a difference. I remember when we were trying to do EPA 535 that if we had any scratches what so ever in the Zymark tubes we would lose 50% of some of the analytes just during blowdown. I have another compound we work with that when stored in a Pyrex 10ml volumetric it will last for a year, but when stored in a plain glass 2ml autosampler vial it will go bad within a week.



If you are looking at analytes that are highly sensitive to pH change, you want to avoid soda lime glass. Always use borosilicate like Pyrex or Kimax. A good example is chlorophenoxyacetic acid herbicides. Recoveries will drop if it spends any time in soda lime glass or if you use soda lime glass boiling beads. Quaternary amines like paraquat and diquat will bond with glass and not come off.

Don
Don Shelly
LGC Standards
Don Shelly wrote:


If you are looking at analytes that are highly sensitive to pH change, you want to avoid soda lime glass. Always use borosilicate like Pyrex or Kimax. A good example is chlorophenoxyacetic acid herbicides. Recoveries will drop if it spends any time in soda lime glass or if you use soda lime glass boiling beads. Quaternary amines like paraquat and diquat will bond with glass and not come off.

Don


Would soda lime glass have enough affinity for PAHs to possibly pull them out of Ethyl Acetate? I doubt it would DCM, but with Ethyl Acetate could it be possible for what is happening above? Especially since one responder mentioned no problems when using silanized glass vials.
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
Don Shelly wrote:


If you are looking at analytes that are highly sensitive to pH change, you want to avoid soda lime glass. Always use borosilicate like Pyrex or Kimax. A good example is chlorophenoxyacetic acid herbicides. Recoveries will drop if it spends any time in soda lime glass or if you use soda lime glass boiling beads. Quaternary amines like paraquat and diquat will bond with glass and not come off.

Don


Would soda lime glass have enough affinity for PAHs to possibly pull them out of Ethyl Acetate? I doubt it would DCM, but with Ethyl Acetate could it be possible for what is happening above? Especially since one responder mentioned no problems when using silanized glass vials.


I don't believe that most PAHs would have a greater affinity to one glass over another (the exception might be polar PAHs). If the problem with the ethyl acetate is enzymatic, then the enzyme would work better in a basic environment. Soda lime glass has a pH around 9.
Don Shelly
LGC Standards
first of all I don't want to take over this tread and I'm willing to put my problem on hold untill the original problem is solved 8)

What procedure do you use to make the calibration standards in DCM? What solvent is the stock standard made in?

Does your extraction involve separatory funnels? What are they made of? What is your spiking technique? What solvent is your spike in?

Yes, PAHs love glass, but their love of solvent is greater.

Don


Our extraction procedure:

100ml sample in duran 150ml GL45 amber bottle
+ 1µl -> 500µl kalibration standard
+ 25µl internal standard (used to determine loss of analyte during extraction)
both standards are made in acetone (BAKER organic residue grade) and spiked with an Eppendorf multipette stream. They are always spiked IN the sample.
+ MgSO4
+ 6 ml DCM
5 min on a linear shaker
DCM is removed with a pyrex pipet (single use off course) and transferd into a test tube (borosil. glass)
Na2SO4 is added to dry
vortex
centrifuge
sample is transfered into a vial that contains a surrugate standard to follow injection reproducibility. transfer is made with 1.250µl colourless eppendorf tips

for our external standards we take a borosil. glass test tube with 4.5ml of DCM (equals the amout of DCM that is recovered from the 6ml added when performing extraction)
spike it with calibration standard and internal standard.
transfer to vial.

so extracted and external standards are made in the same DCM, are made from the same stock-standards, and handled with the same equipment. The only difference is the extraction. We use the difference in external vs.extracted to determine the extraction efficiency.

Peak shape of both extract and external is similar so I don't think the spiking solvent is causing problems in the PTV injection (50µl)

stange thing is that the external comes out low, where you would expect loss of analyte during extraction.

thanks for helping.
BMU
BMU_VMW wrote:
first of all I don't want to take over this tread and I'm willing to put my problem on hold untill the original problem is solved 8)

What procedure do you use to make the calibration standards in DCM? What solvent is the stock standard made in?

Does your extraction involve separatory funnels? What are they made of? What is your spiking technique? What solvent is your spike in?

Yes, PAHs love glass, but their love of solvent is greater.

Don


Our extraction procedure:

100ml sample in duran 150ml GL45 amber bottle
+ 1µl -> 500µl kalibration standard
+ 25µl internal standard (used to determine loss of analyte during extraction)
both standards are made in acetone (BAKER organic residue grade) and spiked with an Eppendorf multipette stream. They are always spiked IN the sample.
+ MgSO4
+ 6 ml DCM
5 min on a linear shaker
DCM is removed with a pyrex pipet (single use off course) and transferd into a test tube (borosil. glass)
Na2SO4 is added to dry
vortex
centrifuge
sample is transfered into a vial that contains a surrugate standard to follow injection reproducibility. transfer is made with 1.250µl colourless eppendorf tips

for our external standards we take a borosil. glass test tube with 4.5ml of DCM (equals the amout of DCM that is recovered from the 6ml added when performing extraction)
spike it with calibration standard and internal standard.
transfer to vial.

so extracted and external standards are made in the same DCM, are made from the same stock-standards, and handled with the same equipment. The only difference is the extraction. We use the difference in external vs.extracted to determine the extraction efficiency.

Peak shape of both extract and external is similar so I don't think the spiking solvent is causing problems in the PTV injection (50µl)

stange thing is that the external comes out low, where you would expect loss of analyte during extraction.

thanks for helping.
BMU



I think that the next course of action would be to have a colleague QC the external standard dilution procedure.

You probably do not need the magnesium sulfate in your extraction procedure.
Don Shelly
LGC Standards
You probably do not need the magnesium sulfate in your extraction procedure.


By adding MgSO4 we make it al lot easier to recover the DCM from the water, since it lowers solubility quit a bit. In most cases this isn't necessary but when analysing surface water it is necessary to add MgSO4.
From what I've been reading the method in question is not 525.2, or am I mistaken? The way I read 525.2 it is a liquid solid extraction method.
Bigbear wrote:
From what I've been reading the method in question is not 525.2, or am I mistaken? The way I read 525.2 it is a liquid solid extraction method.


You are correct. BMU apologized for entering the discussion a few entries back.

That phrase brings up a pet-peeve of mine. Nothing personal. :)
525.2 refers to this method as solid-liquid. 525.3 refers to the method as solid-phase extraction. EPA corrected the mistake they had made in previous method titles. Solid-liquid refers to a diatomaceous earth column where the liquid matrix is dispersed over a solid matrix and then extracted using a solvent. In solid-phase extraction, the matrix passes through the cartridge and is removed. The analytes bond/bind to the sorbent until acted upon by a strong solvent.

Ahhh. I feel better now. :lol:
Don Shelly
LGC Standards
Was anyone able to come to a conclusion about the loss of PAHs?

I am having the same problem with my SUR Benzo[a]pyrene-d12 in EPA 525.3. For all of my extracted samples Benzo[a]pyrene-d12 come out to 50-65% recovery.

I have tried traditional and matrix matched calibration standards and they are almost identical.

I have tried extracting these in the dark- no improvement
I used a better quality Methanol- no improvement
The matrix match cal std use the same solvents so I don't expect those to be the culprit

All samples/std are stored in the same freezer.

I am following the EPA method as instructed for DVB disks and I cannot understand why my Benzo[a]pyrene-d12 SUR recovery is consistently low?

Any help would be appreciated, thank you!!
LWC wrote:
Was anyone able to come to a conclusion about the loss of PAHs?

I am having the same problem with my SUR Benzo[a]pyrene-d12 in EPA 525.3. For all of my extracted samples Benzo[a]pyrene-d12 come out to 50-65% recovery.

I have tried traditional and matrix matched calibration standards and they are almost identical.

I have tried extracting these in the dark- no improvement
I used a better quality Methanol- no improvement
The matrix match cal std use the same solvents so I don't expect those to be the culprit

All samples/std are stored in the same freezer.

I am following the EPA method as instructed for DVB disks and I cannot understand why my Benzo[a]pyrene-d12 SUR recovery is consistently low?

Any help would be appreciated, thank you!!


Unless you are using an Empore disk, or a cartridge that uses Teflon frits, you are probably losing the d12 to either glass wool in a disk or polyethylene frits in a cartridge. I have no explanation of how this can happen, but it happens.

What product are you using?
Don Shelly
LGC Standards
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