Limit of nitrilotriacetic acid in EDTA by USP method

[Murshad]

Hi Fellows

I have problem

I am doing Limit of nitrilotriacetic acid in EDTA by USP method

M Phase:
10ml of 1.0M tetrabutylammonium hydroxide in methanol in 200ml water, pH adjusted to 7.5 with 1M phosphoric acid, transferred to 1000ml vol flask added 90ml methanol diluted to vol with water. Mixed, filtered through 0.45um filter and degassed.

Cupric nitrate sol:
500 mg of cupric nitrate in 500ml of water

Standard stock solution:
100mg of nitrilotriacetic acid + 0.5ml ammoniun hydroxide diluted to vol in 10ml vol flask

Resolution standard:
10mg EDTA+100ul standard stock solution diluted to 100ml with cupric nitrate solution

Standard solution:
1.0g EDTA + 100ul standard stock solution diluted to 100ml with cupric nitrate solution

Sample solution:
1.0g EDTA diluted to 100ml with cupric nitrate solution

Column is 150mm x 4.0mm L7
Wavelength 254nm
Injection vol 50ul
Col temp 25°C

My problem is inconsistency in response

Even when in injected resolution standard it was not possible to get consistence response for contineous 3 chromatograms, peaks are distorted and I got good chromatogram after a every 5,6 injections.
I have also tried loop wash but that did not work/
Please omit all possibilities with Chromatograph. My guess is something happening in column

Thanks with regards

[Mark Tracy]

Are the 5,6,7,8,... injections consistent? If so, you just need to have adequate equilibration time. Also, there may be metal binding sites on the column that need to be saturated before you get consistent response. Try rapid-fire injections of 6x50µL of your standard as part of the warmup.