HPLC Column Companion, Section 3: What is the Best Column Diameter for Your Method?


Section 3 What is the Best Column Configuration for Your Method?

The availability of analytical HPLC columns with internal diameters of less than 4.6 mm provides the chromatographer with options for developing more cost efficient methods. Solvent Saver™ (3.0 mm i.d.) and narrow-bore (2.1 mm i.d.) columns provide several important benefits for chromatographers. These include:

	Reduced Mobile Phase Expenses - less solvent usage - lower solvent disposal costs
	Enhanced Sensitivity for Mass-Limited Samples - Solvent Saver columns can increase sensitivity by a factor of 2 - Narrow-Bore columns can increase sensitivity by a factor of 5
	Reduced Sample Size Requirements - smaller sample sizes can be used effectively - sample preparation requires smaller volumes of reagents
	Increased Compatibility with Special Detectors - LC/MS - LC/NMR

These potential benefits are all a result of the reduced internal column volume of smaller i.d. columns.

One way to reduce solvent consumption in HPLC is to use columns with a smaller internal diameter than "standard" 4.6 mm i.d. columns Solvent Saver and narrow-bore columns can reduce solvent consumption by 60-80%. Table 3-1 shows the relationship between column internal diameter, column volume, and solvent waste reduction.

Table 3-1

Column Dimensions mm	Internal Volume mL	Calculated Peak Volume 無	Solvent Consumption mL	N	% Solvent Reduction

4.6 x 250	2.50	550	18.9	19,000	0
4.6 x 150	1.50	430	11.4	11,000	40
3.0 x 250	1.06	240	8.0	19,000	58
3.0 x 150	0.64	188	4.8	11,000	74
2.1 x 150	0.30	94	2.4	11,000	87

NOTE: k of last eluting peak is 6.6

Reduced column internal diameters result in smaller column volumes. It is this reduction in column volume that allows for an increase in detection limits.When the column volume is decreased the peak volume is decreased (Table 3-1). This means that when the same sample volume is injected on a 4.6 mm i.d. column and a 3.0 mm i.d. column, the peak volume will be smaller on the 3.0 mm i.d. column. The same sample is more concentrated in the smaller peak volume and the result is enhanced sensitivity. [NOTE: Be sure to read the discussion on Extra-Column Volume - Section 4]

Optimum Efficiency with Small-Bore Columns is Directly Related to Peak Volume

Optimum efficiency (N) requires that your HPLC be compatible with small-bore columns. The peak volume of each eluting band decreases significantly as the diameter of the column is decreased (Table 3-1). Peaks with low k values are especially critical, as they have the lowest peak volume in the chromatogram.

A well-designed standard HPLC system may tolerate column peak volumes as low as 80 無 and still provide excellent efficiency. However, peak volumes below 80 無 will require careful system design. Standard 4.6 mm i.d. and 3.0 mm i.d. Solvent Saver columns can be used on most HPLC systems with optimum efficiency, but 2.1 mm i.d. narrow-bore columns may require HPLC system optimization to be used effectively (see Section 4, Correcting Excessive Extra Column Volume). Columns below the 2.1 mm i.d. size require sophisticated HPLC hardware systems not commonly found in today's laboratories.

Many chromatographers use columns of reduced column diameter (< 4.6 mm i.d.) to reduce solvent waste or increase sensitivity when sample is limited. No matter what your goal is for switching to a reduced diameter column, it is important to know that a simple column substitution without modification of flow rate will compromise resolution. Substituting a column having the same packing, the same length, but a smaller diameter requires that you reduce the flow rate in order to retain the same retention time and resolution for peaks in the original chromatogram.

Figure 3-2
Separation of Nitrobenzenes on Columns of Different Diameters
with the Same Flow Rate

Figure 3-2 illustrates what you can expect if you substitute columns of smaller inner diameter without reducing now rate. In this example, the same sample is separated using identical experimental conditions on columns having 4.6 mm, 3.0 mm and 2.1 mm inner diameter (i.d.).The flow rate for each separation is 1.0 ml/min.The resultant chromatograms are very different.The retention and critical resolution between peak pair 2,3 on the 4.6 mm i.d. column decreases significantly on the 3.0 mm i.d. column and even more so on the 2.1 mm i.d. column.

As the column diameter decreases, the column void volume (V_m) decreases. The column void volume for the three columns used in Figure 3-3 is listed in the Table below.

Table 3-2
Changing Column Diameter Requires Flow Rate Adjustment

Column	Column Void Volume (mL)	Relative Flow Rate (mL/min)	k = [t_r - (V_m / F)] / (V_m / F)

4.6 x 150 mm	1.5	1.0
3.0 x 150 mm	0.6	0.4
2.1 x 150 mm	0.3	0.2

If the mobile phase and stationary phase is unchanged, k is unchanged. To maintain the same retention time (t_r), the flow rate (F) must decrease to compensate for a decrease in column void volume (V_m).

For example:
V_m1 / F₁ = V_m2/ F₂ ; 1.5 / 1.0 = 0.6 / F₂
1.5 F₂ = 0.6
F₂ = 0.4 mL/min

To keep retention time unchanged, the ratio V_m/F must be held constant for each separation. Therefore, a decrease in V_m, must be followed with a proportional decrease in F (see Table 3-2 for example calculation). Figure 3-3 shows that running the 3.0 and 2.1 mm i.d. columns at 0.4 mL/min and 0.2 mL/min respectively, retrieves the original separation with the promised reduction in solvent usage.

Figure 3-3
Separation of Nitrobenzenes on Columns of Different Diameters with the Adjusted Flow Rate