Regis Technologies

Ion Pair Reagents

Ultra pure Ion pair reagents and buffers

 
How to Select a Regis Ion Pair Reagent for Method Development?
To choose the proper ion pair reagent, alkyl chain lengths must be taken into consideration. The chain lengths enable selective separation of the analyte. The longer the chain, the more hydrophobic the counter-ion, therefore, the greater the retention. Retention may increase by a factor of almost 20 when going from pentyl (Q5) to dodecyl (Q12), as illustrated in Table 1 and Figure 1.
 
 
Retention Times
(min)
Retention Times
(min)
Retention Time
Ratio

Q - Series

Benzoic Acid

Benzyl Alcohol

Acid/Alcohol

Q5

4.53

9.17

0.49

Q6

6.50

8.60

0.76

Q7

8.25

9.13

0.90

Q8

12.36

8.94

1.38

Q12

79.53

8.52

9.33
Table 1. Retention vs. Chain Length
 
 
Key:
III = benzoate
A = benzyl alcohol
 
Column:
Rexchrom ODS 15 cm x 4.6 mm i.d., fully endcapped
 
Mobile Phase:
(60/40) water/methanol + 0.005 M Q-Series
 
Flow Rate:
0.6 mL/min
Figure 1. Effect of Q-Reagent Chain Length on Retention 
 
Both Table 1 and Figure 1 demonstrate that the Q-reagent chain length governs benzoic acid retention times, but does not affect the benzyl alcohol retention times. Similar behavior can also be achieved with the S-Series.
 
Guidelines for developing a successful method using Regis ion pair reagents:
  1. Select a column - endcapped ODS (octadecylsilyl) is most common.
  2. Use only HPLC-grade water and chromatography grade reagents in mobile phase preparation.
  3. Choose the mobile phase components and concentrations that give the best separation.
  4. If nonionic components are present in the sample, optimize the resolution prior to attempting ionic separations.
  5. Select the appropriate ion pair series to provide the necessary counterion. Using the Q-series for acidic compounds and the S-series for basic compounds.
  6. Through a process of elimination, choose the alkyl chain length which results in the best separation [Figure 2].
Key:
Acids:
I = isophthalic
II = phthalic
III = benzoic
IV = 1-naphthalene sulfonic
Alcohols:
A = benzyl
B = o-methylbenzyl
C = p-methylbenzyl
Column:
Rexchrom ODS 15 cm x 4.6 mm i.d. fully endcapped
Mobile Phase:
(60/40) water/methanol + 0.005 M Q-Series
Flow Rate:
0.6 mL/min
Figure 2. Choosing Appropriate Ion Pair Reagents 
 
In a mixture of ionic and nonionic compounds, first separate the nonionic compounds from each other [See above]. Then choose the ion pair reagent that retains the ionic compounds as desired. Here, Q6 seems to be the reagent of choice since all peaks are visibly separated.
  • Once the reagent has been selected, adjust the pH of the mobile phase to maximize resolution. Because slight modification of pH can profoundly affect retention and selectivity, make all adjustments in small increments and monitor carefully [Table 2].

Q6

Q7

Q8

pH

R

pH

R

pH

R

7.50

0.59

7.50

0.88

7.51

1.06

6.50

0.70

6.51

1.00

6.54

1.29

5.50

0.96

5.52

1.23

5.50

1.59
Table 2. Retention Ratio R as a Function of pH [benzoic acid/benzyl alcohol in (60/40) water/methanol] 
  • Ideally, the ion pair reagent concentration in the mobile phase should be 0.005 M. However, small adjustments in reagent concentration may increase retention slightly and optimize the separation [Figure 3].
Column:
Rexchrom ODS 15 cm x 4.6 mm i.d. fully endcapped
 
Mobile Phase:
(60/40) water/methanol+ Q-Series
 
Flow Rate:
0.6 mL/min
Figure 3. Changing Q Concentration to Fine-Tune a Separation 
 
Increasing Q7 concentration from 0.005 M (left) to 0.0055 M (right) moves the benzoate peak off the benzyl alcohol peak.