Column selection for USP <467> using ProEZGC

There are a lot of decisions to be made when performing USP <467>. With three classes of solvents to potentially monitor, two phase materials to choose from, two dimensions for each phase, and the option to select and validate your column of choice, it can be daunting to start thinking about analysis. In addition, unless you are making reference standards in-house, residual solvent stocks are sub-divided into a series of mixtures to be run individually, rather than one ‘megamix’. This helps improve separations, save time, and make your lab more efficient, because one run with fewer compounds is faster and easier to analyze, but it also means adjusting methodology when it comes time to monitor a new solvent, which can cause a rift in efficiency. This is where Restek’s ProEZGC can help.

Reference standard selection:

The first challenge to overcome is what reference standards are needed. The most up-to-date stock solutions offered at Restek are:

Table 1: USP <467> reference standards available through Restek, color coordinated with Tables 3 and 5.

Cat.#	Mix
36279	Class 1
36015	Class 2 Mix A
36280	Class 2 Mix B
36273	Class 2 Mix C
36016	Class 3 Mix A
36014	Class 3 Mix B

 

These mixes represent changes that have been implemented in chapter <467> as recently as 2020. They were also used to build our ProEZGC libraries using the recommended Rxi-624sil MS (G43) and Stabilwax (G16) phases. 

Column selection:

USP <467> recommends four possible columns, comprised of two phases: G16 and G43, and two dimensions for each phase. The Restek equivalents for these columns are as follows:

Table 2: Restek columns available in recommended USP <467> phases and dimensions.

Cat.#	Mix
10624	(G16) Stabilwax GC Capillary Column, 30 m, 0.32 mm ID, 0.25 µm
10625	(G16) Stabilwax GC Capillary Column, 30 m, 0.53 mm ID, 0.25 µm
13870	(G43) Rxi-624Sil MS GC Capillary Column, 30 m, 0.32 mm ID, 1.80 µm
13871	(G43) Rxi-624Sil MS GC Capillary Column, 30 m, 0.53 mm ID, 3.00 µm

 

To start exploring separations on these phases and dimensions, go to ProEZGC. The simplest way to get started is to copy/paste your list of compounds either by name or CAS#, into the appropriate textbox. You can search mix by mix, or search for all mixes at once. For this example, I have searched for all compounds at once:

The first column recommended is a G16 phase. By going to the stationary phase results dropdown, we see the Rxi-624sil MS is recommended next. So here is where you get to decide how to proceed – you can move forward with either phase! But, for the sake of this article, I moved forward with the Stabilwax.

Figure 1: The search window for ProEZGC. Arrows indicate the solve button, and dropdown of column phases with solvent libraries available for modeling.

The default dimensions recommended did not match the USP <467>, and I was curious why not. So, I went into the conditions tab, selected the recommended dimensions, and optimized some separations for maximum column efficiency. I then compared separations between recommended Stabilwax column dimensions, and the default dimensions offered.

Figure 2: ProEZGC conditions tab with default options. Arrows indicate the conditions tab, column dimensions dropdown menu, USP <467> recommended column dimensions, oven refinement and optimization button, and radio button to optimize by efficiency.

When comparing the separations across columns, I primarily looked at separations within mixes. While there were many co-elutions between mixes, those can be easily separated by running just one mix at a time. The separations within mixes offer a more complex challenge. 

Below is a table showing the predicted separations across different Stabilwax (G16) columns, where co-elutions within mixes are boxed in red. Different colored rows represent different mixes, and red-cells highlight all co-elutions determined by ProEZGC. After optimization, the recommended column dimensions, cat.# 10624 and 10625, each had four within-mix co-elutions, while the default recommendation by ProEZGC (cat.# 10654) had only two.

Table 3: Comparison of G16 column dimensions by co-elution. Colors represent different mixes (see Table 1), red represents co-elutions, red-boxes represent within-mix co-elutions.

#	Stabilwax Compound	Stabilwax cat.# 10624		Stabilwax cat.# 10625		Stabilwax cat.# 10654
		Compound	RT	Res	RT	Res	RT
1	n-Pentane	1.74	4	2.36	1.9	1.86	14.4
2	C6	1.82	2.4	2.43	1.2	2.18	7.4
3	Diethyl ether	1.87	2.4	2.47	1.2	2.37	7.4
4	Methyl-tert-butyl ether	1.99	1.2	2.57	0.6	2.84	2.9
5	n-Heptane	2.01	1.2	2.59	0.6	2.94	2.9
6	Cyclohexane	2.1	2.4	2.66	1.2	3.28	5.3
7	1,1-Dichloroethene	2.15	2.4	2.71	1.2	3.5	5.3
8	Methylcyclohexane	2.31	6.7	2.88	3.5	4.13	13.9
9	Acetone	2.65	4.4	3.12	2.5	5.5	7.1
10	Ethyl formate	2.79	0.3	3.23	0.1	6.02	0.4
11	Methyl acetate	2.79	0.3	3.24	0.1	6.06	0.4
12	Tetrahydrofuran	3.05	1.3	3.45	0.7	7.03	1.6
13	Triethylamine	3.09	1.3	3.48	0.7	7.17	1.6
14	trans-1,2-Dichloroethene	3.35	0.4	3.7	0.3	7.95	1.2
15	1,1,1-Trichloroethane	3.37	0.1	3.71	0.1	8.05	0.1
16	Carbon tetrachloride	3.38	0.1	3.72	0.1	8.07	0.1
17	Ethyl acetate	3.49	2.9	3.81	1.8	8.37	3.7
18	Methanol	3.66	1.9	3.99	1.1	8.79	2.5
19	2-Butanone	3.75	1.9	4.03	1.1	8.98	2.5
20	Isopropyl acetate	4.12	0.1	4.33	--	9.51	3.5
21	1,2-Dimethoxyethane	4.12	0.1	4.34	--	9.75	3.3
22	2-Propanol	4.29	1.3	4.48	0.8	10	1.9
23	Methylene chloride	4.36	1.3	4.54	0.8	10.14	1.9
24	Benzene	4.48	2.2	4.63	1.4	10.39	3.3
25	Ethanol	4.81	6.1	4.91	4	10.81	5.7
26	Propyl acetate	5.59	6.3	5.56	4.5	11.82	5.8
27	Trichloroethene	6.02	5.3	5.94	3.7	12.23	5.7
28	Acetonitrile	6.38	1.3	6.27	0.9	12.57	1.5
29	4-Methyl-2-pentanone	6.47	1.3	6.35	0.9	12.65	1.5
30	Isobutyl acetate	6.7	3.3	6.55	2.3	12.77	2.2
31	Chloroform	6.94	2.3	6.76	1.7	12.91	0.6
32	2-Butanol	7.1	2.3	6.9	1.7	12.94	0.6
33	Toluene	7.34	1.9	7.09	1.5	13.15	4
34	1-Propanol	7.47	1.9	7.2	1.5	13.32	4
35	1,4-Dioxane	7.96	7.3	7.58	5.2	13.66	3.2
36	1,2-Dichloroethane	8.44	--	7.94	--	13.79	--
37	Butyl acetate	8.45	--	7.95	--	13.8	--
38	2-Hexanone	8.64	3	8.09	2.1	13.95	2.1
39	Isobutanol	9.12	7.5	8.43	5.4	14.03	2.1
40	Ethylbenzene	9.95	3.5	9	2.5	14.65	1.5
41	p-Xylene	10.17	2.8	9.14	2	14.7	1.5
42	m-Xylene	10.35	2.8	9.26	2	14.75	1.5
43	1-Butanol	10.66	4.9	9.45	3.4	14.83	2.2
44	Nitromethane	11.01	4.3	9.66	3	14.95	3.5
45	Isopropylbenzene	11.26	2.2	9.81	1.5	15.17	0.1
46	Pyridine	11.39	1.5	9.88	0.9	15.17	0.1
47	2-Methoxyethanol	11.48	1.5	9.93	0.9	15.26	2.7
48	3-Methyl-1-butanol	12.19	2	10.31	1.6	15.35	2.8
49	Chlorobenzene	12.28	2	10.38	1.6	15.55	2.5
50	2-Ethoxyethanol	12.39	2.5	10.44	1.6	15.63	2.5
51	1-Pentanol	12.9	12.5	10.78	8.7	15.72	3
52	N,N-Dimethylformamide	13.83	4.9	11.46	3.2	16.6	2.4
53	Anisole	13.97	4.9	11.57	3.2	16.67	2.4
54	N,N-Dimethylacetamide	14.49	17.6	11.97	11.8	17.13	5.3
55	Acetic acid	14.95	17.6	12.34	11.5	17.29	5.3
56	Tetralin	15.36	9.9	12.66	6.6	17.93	4.1
57	Dimethyl sulfoxide	15.6	1.6	12.86	1.5	18.04	4.1
58	Formic acid	15.64	1.6	12.9	1.5	18.22	1.5
59	Ethylene glycol	15.82	8.8	13.04	5.4	18.27	1.5
60	N-Methylpyrrolidone	16.16	15.5	13.3	9.5	18.9	9
61	Formamide	16.6	18.5	13.67	12.8	19.17	9
62	Sulfolane	16.65	94.8	15.35	62.6	24.18	168

 

The optimized GC conditions for each column are as follows:

Table 4: GC Conditions for columns in Table 3.

Column:	Stabilwax, 30.00 m, 0.32 mm ID, 0.25 µm (cat.# 10624)
Carrier Gas:	Helium, Constant Flow @ 1.79 mL/min
Average Velocity:	29.48 cm/sec
Outlet Pressure (abs):	14.70 psi
Oven Temp.:	30 °C (hold 5.5 min) to 70 °C @ 6.5 °C/min to 225 °C @ 26 °C/min (hold 3 min)
Column:	Stabilwax, 30.00 m, 0.53 mm ID, 0.25 µm (cat.# 10625)
Carrier Gas:	Helium, Constant Flow @ 2.97 mL/min
Average Velocity:	21.48 cm/sec
Outlet Pressure (abs):	14.70 psi
Oven Temp.:	30 °C (hold 6 min) to 75 °C @ 13.5 °C/min to 225 °C @ 30 °C/min (hold 3 min)
Column:	Stabilwax, 30.00 m, 0.32 mm ID, 1.00 µm (cat.# 10654)
Carrier Gas:	Helium, Constant Flow @ 1.79 mL/min
Average Velocity:	29.65 cm/sec
Outlet Pressure (abs):	14.70 psi
Oven Temp.:	30 °C (hold 6.5 min) to 65 °C @ 6.5 °C/min to 225 °C @ 23 °C/min (hold 7 min)

 

I repeated this experiment for the Rxi-624sil MS to preview what separations I would encounter on the alternative set of phases and dimensions. Below is a table showing the predicted separations across different columns, this time using the Rxi-624sil MS phase. Different colored rows represent different mixes, and red-cells highlight all co-elutions determined by ProEZGC. After optimization, the recommended column dimension cat.# 13870 has two within-mix co-elutions, and cat.# 13871 had four. The default recommendation by ProEZGC (cat.# 13868) also only had two within-mix co-elutions, but provided improved separation of isopropyl acetate and m-xylene relative to cat.# 13870.

Table 5: Comparison of G43 column dimensions by co-elution. Colors represent different mixes (see Table 1), red represents co-elutions, red-boxes represent within-mix co-elutions.

#	Rxi-624sil Compound	Stabilwax cat.# 13870		Stabilwax cat.# 13871		Stabilwax cat.# 13868
		RT	Res	RT	Res	RT	Res
1	Ethylbenzene	2.48	22.9	3.22	15.7	2.39	28
2	Anisole	3.33	4.6	4.33	3.1	3.21	5.5
3	p-Xylene	3.57	2.4	4.64	1.6	3.44	2.9
4	1,2-Dichloroethane	3.7	2.4	4.81	1.6	3.56	2.9
5	Ethylene glycol	4.06	2.1	5.28	1.5	3.92	2.6
6	4-Methyl-2-pentanone	4.2	2.1	5.46	1.5	4.05	2.6
7	Isopropyl acetate	4.52	1.2	5.88	0.8	4.35	1.4
8	m-Xylene	4.6	1.2	5.98	0.8	4.43	1.4
9	Methylcyclohexane	4.77	2.3	6.2	1.6	4.59	2.8
10	Toluene	4.96	2.4	6.46	1.7	4.78	2.9
11	Chlorobenzene	5.15	2.4	6.7	1.7	4.96	2.9
12	Propyl acetate	5.8	0.3	7.47	0.2	5.59	0.4
13	n-Pentane	5.84	0.3	7.5	0.2	5.62	0.4
14	2-Methoxyethanol	6.57	8.2	8.06	4.9	6.29	8.4
15	Ethyl formate	7.39	6.8	8.55	5.2	6.84	8.4
16	Tetrahydrofuran	7.94	2.7	9.01	1.9	7.28	3.2
17	Isobutyl acetate	8.12	1.7	9.17	0.9	7.43	1.8
18	C6	8.22	1.5	9.24	0.8	7.51	1.6
19	1,2-Dimethoxyethane	8.32	1.5	9.31	0.8	7.58	1.6
20	2-Ethoxyethanol	8.52	1.9	9.53	0.6	7.78	1.7
21	Cyclohexane	8.66	0.5	9.58	0.6	7.85	0.9
22	Pyridine	8.66	0.5	9.64	0.8	7.89	0.9
23	Tetralin	8.84	1.4	9.84	1	8.07	1.7
24	Triethylamine	8.94	1.4	9.92	1	8.15	1.7
25	3-Methyl-1-butanol	9.06	2.2	10.02	1.4	8.25	2.5
26	Butyl acetate	9.29	0.1	10.22	0.3	8.46	0.2
27	1,4-Dioxane	9.3	0.1	10.24	--	8.47	0.2
28	Sulfolane	9.33	0.4	10.24	--	8.49	0.3
29	N,N-Dimethylacetamide	9.35	0.3	10.25	--	8.5	0.3
30	Ethyl acetate	9.37	0.3	10.3	0.7	8.53	0.7
31	n-Heptane	9.46	1.7	10.36	0.7	8.61	1.9
32	cis-1,2-Dicholoroethene	9.54	1.7	10.46	1.5	8.69	2.2
33	trans-1,2-Dichloroethene	9.7	3.5	10.61	2.2	8.84	3.9
34	Methyl-tert-butyl ether	10.02	0.4	10.9	0.9	9.13	0.9
35	Carbon tetrachloride	10.04	0.4	10.96	0.9	9.16	0.9
36	2-Hexanone	10.28	2.1	11.21	1.1	9.39	2.3
37	Diethyl ether	10.37	1.8	11.29	0.8	9.47	1.9
38	Ethanol	10.45	1.8	11.34	0.8	9.54	1.9
39	Methanol	10.68	5.9	11.6	4.1	9.77	7
40	2-Propanol	11.05	0.7	11.98	0.8	10.13	1
41	Acetone	11.08	0.7	12.03	0.4	10.17	1
42	Chloroform	11.14	1.5	12.06	0.4	10.22	1.5
43	1-Propanol	11.22	0.6	12.16	0.3	10.31	0.4
44	1-Butanol	11.25	0.6	12.19	0.3	10.32	0.4
45	1-Pentanol	11.34	2.5	12.28	1.5	10.42	3.1
46	Benzene	11.55	5.4	12.5	3.6	10.62	6.5
47	1,1,1-Trichloroethane	11.75	2.6	12.72	1.6	10.83	3.1
48	Acetonitrile	11.85	2.6	12.82	1.6	10.92	3.1
49	Methylene chloride	12	4.1	12.98	2.5	11.07	4.9
50	Formamide	12.39	1.8	13.44	1.1	11.47	2.1
51	1,1-Dichloroethene	12.46	1.8	13.51	1.1	11.55	2.1
52	Nitromethane	12.56	--	13.62	--	11.65	--
53	Isobutanol	12.56	--	13.62	--	11.65	--
54	2-Butanol	13.07	1.8	14.18	1.3	12.17	2.2
55	2-Butanone	13.14	1.7	14.26	1.1	12.24	2
56	Trichloroethene	13.2	1.7	14.33	1.1	12.3	2
57	Methyl acetate	14.81	18.4	16.14	12.6	13.96	22.3
58	N-Methylpyrrolidone	15.49	18.4	16.92	12.6	14.66	22.3
59	Isopropylbenzene	16.59	28.9	18.17	19.7	15.8	34.6

 

The optimized GC conditions for each column are as follows:

Table 6: GC Conditions for columns in Table 5.

Column:	Stabilwax, 30.00 m, 0.32 mm ID, 0.25 µm (cat.# 10624)
Carrier Gas:	Helium, Constant Flow @ 1.79 mL/min
Average Velocity:	29.48 cm/sec
Outlet Pressure (abs):	14.70 psi
Oven Temp.:	30 °C (hold 5.5 min) to 70 °C @ 6.5 °C/min to 225 °C @ 26 °C/min (hold 3 min)
Column:	Stabilwax, 30.00 m, 0.53 mm ID, 0.25 µm (cat.# 10625)
Carrier Gas:	Helium, Constant Flow @ 2.97 mL/min
Average Velocity:	21.48 cm/sec
Outlet Pressure (abs):	14.70 psi
Oven Temp.:	30 °C (hold 6 min) to 75 °C @ 13.5 °C/min to 225 °C @ 30 °C/min (hold 3 min)
Column:	Stabilwax, 30.00 m, 0.32 mm ID, 1.00 µm (cat.# 10654)
Carrier Gas:	Helium, Constant Flow @ 1.79 mL/min
Average Velocity:	29.65 cm/sec
Outlet Pressure (abs):	14.70 psi
Oven Temp.:	30 °C (hold 6.5 min) to 65 °C @ 6.5 °C/min to 225 °C @ 23 °C/min (hold 7 min)

 

Overall, this comparison provided some important insight into the separations to expect when changing column dimensions. The ProEZGC default dimensions certainly seemed to provide better solutions then the USP <467> recommended dimensions for both phases. Depending on your analysis, this might save you a significant amount of trial-and-error trying to determine which of the suggested columns will work best.

If we choose to compare instead between the phases, using results from the ProEZGC recommended phases, we see that each column results in two within-mix co-elutions. Using the Stabilwax, we can expect ethyl formate and methyl acetate from Class 3 Mix A, and 1,1,1-Trichloroethane and carbon tetrachloride from Class 1 mix, to co-elute. Using the Rxi-624sil MS, we can expect isopropyl acetate and m-xylene from Class 3 Mix A, and nitromethane and isobutanol from Class 2 Mix A, to co-elute. All other co-elutions can be overcome by running one mix at a time. Depending on which solvents are more relevant to your lab, either column may be ideal.

Conclusions:

With so many options to sift through, and so many opportunities to tailor your lab efficiency, implementing USP <467> can be complicated. With the help of ProEZGC, Restek can helps your lab get running as fast as possible, and with as little troubleshooting as possible, as you build GC methods that match your labs needs.